Metastasis and the Impact of Translation in the Tumor and Host: a Phospho-Eif4e Story

Metastasis and the Impact of Translation in the Tumor and Host: a phospho-eIF4E Story

by Nathaniel Robichaud

Department of Biochemistry Rosalind and Morris Goodman Cancer Research Centre McGill University Montréal, Québec, Canada

December 2017

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy.

©Nathaniel Robichaud, 2017

TABLE OF CONTENTS Title page…………………………………………………………………………………………………………………………………i Table of contents…………………………………………………………………………………………………………………….ii English abstract……………………………………………………………………………………………………………………vii French abstract……………………………………………………………………………………………………………………viii Preface and contributions of authors……………………………………………………………………………………ix Contributions to knowledge and elements of original scholarship…………………………………………xi Acknowledgements………………………………………………………………………………………………………………xiii Chapter 1: General Introduction…………………………………………………………………………………………1 Rational and objectives…………………………………………………………………………………………………………..2 1. Literature review…………………………………………………………………………………………………………………2 1.1 On the importance of translational control in cancer…………………………………………………………2 1.2 Brief overview of translational control……………………………………………………………………4 1.3 eIF4E binds the cap…………………………………………………………………………………………………7 1.4 Regulation of eIF4E..….…………………………………………………………………………………………10 1.4.1 eIF4E interacting proteins: eIF4G……….………………………………………………….10 1.4.2 eIF4E interacting proteins: 4E-BPs..……..………………………………………………..10 1.4.3 eIF4E phosphorylation.………………………………………………………………………….11 1.5 Deregulated translation in cancer……………………………………………………………………………………12 1.5.1 Specific effects of general translation initiation factors……………………………………..12 1.5.2 Mechanisms of deregulated translation in cancer …………………………………………….13 1.5.2.1 Changes in cis: 5’ and 3’UTR length and composition………..……………….13 1.5.2.2 Oncogenic signaling…………….……………………………………………………………..17 1.5.2.3 Gain/loss of initiation factors.…………………………………………………………….21 1.5.2.3.1 eIF4F complex formation……………………………………………………..21 1.5.2.3.2 Pre-initiation complex formation…….……………………………………23 1.5.2.3.3 eIF3, connecting the eIF4F and pre-initiation complexes……..24 1.5.2.4 Alternative initiation…….…………………………………………………………………….25 1.5.2.5 Translation elongation and termination.…………………………………………….25

1.5.2.5 Factors regulating multiple steps of translation..………………………………..26 1.5.2.5.1 eIF6 and eIF5A…………………………………….………………………………..26 1.5.2.5.2 The MYC oncoprotein…………………………………………………………..27 1.5.2.5.3 The cancer ribosome………………….…………………………………………28 1.5.2.6 A variety of initiation factors with a variety of effects………………………….29 1.5.3 Selective advantages of deregulated translation……………..……………………………….30 1.5.3.1 Proliferation and apoptosis……………………………………..………………………….30 1.5.3.2 Angiogenesis………………………………………………………….…………………………..30 1.5.3.3 Stress response.………………………………………………………………………………….31 1.5.3.4 Oncogene Induced Senescence (OIS).…………………………………………………32 1.5.3.5 Metabolism…….……………………………….…………………………………………………33 1.5.3.6 Drug resistance………………………………….……………………………………………….34 1.5.3.7 Emerging advantages of deregulated translation………….……………………35 1.5.4 Inhibitors of translation……….……………………………………………………………………………35 1.5.4.1 mTOR inhibitors - Rapamycin and its analogs……………………………………..35 1.5.4.2 Second-generation mTOR inhibitors……………………………………………………36 1.5.4.3 MNK inhibitors……………………………………………………………………………………38 1.5.4.4 Inhibitors of eIF4E-cap interaction……….…………………………………………….38 1.5.4.5 Inhibitors of eIF4E-eIF4G interaction……..……………………………………………39 1.5.4.6 Inhibitors of eIF4A………………………………….…………………………………………..40 1.5.4.7 Inhibitors of ternary complex formation..……………………………………………41 1.5.4.8 Other inhibitors of translation……………………..……………………………………..41 1.5.5 Context specificity……………………………………………………..……………………………………..42 1.5.5.1 Cancer cell stemness.………………………………………………………………………….43 1.5.5.2 Fibroblasts……………………………………….………………………………………………...44 1.5.5.3 Immune cells………………………………………………………………………………………44 1.6 Concluding remarks…………………………………………………………………………………………………………45 Chapter 2: Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3…………………………………………………………………………………………………………46

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2.1 Contextual setting…………………………………………………………………………………………………………..47 2.2 Results…………………………………………………………………………………………………………………………….49 2.2.1 eIF4E phosphorylation promotes migration and invasion………………………………….49 2.2.2 Phospho-eIF4E promotes the translation of metastasis-related mRNAs……………52 2.2.3 eIF4E is phosphorylated during TGFβ-induced EMT…………………………………………..58 2.2.4 eIF4E phosphorylation promotes TGFβ-induced EMT……………………………………….61 2.2.5 eIF4E phosphorylation correlates with EMT and invasion in vivo………………………62 2.2.6 eIF4E phosphorylation promotes tumor onset and metastasis in PyMT mammary tumor model………………………………………………………………………………………………………..……65 2.3 Discussion……………………………………………………………………………………………………………………….70 2.4 Materials and Methods……………………………………………………………………………………………………71 2.5 Acknowledgments…………………………………………………………………………………………………………..75 Chapter 3: Translational Control in the Tumor Microenvironment Promotes Lung Metastasis via Phosphorylation of eIF4E in Neutrophils………………………………………………………………………76 3.1 Contextual setting……………………………………………………………………………………………………………77 3.2 Results…………………………………………………………………………………………………………………………….79 3.2.1 Phosphorylation of eIF4E in breast cancer patients…..……………………………………..79 3.2.2 Phosphorylation of eIF4E in the TME promotes metastasis in vivo…………………..81 3.2.3 Excluding potential mediators of the effect of phospho-eIF4E in the TME………..84 3.2.4 Neutrophils promote metastasis……………………………………………………………………….85 3.2.5 Phospho-eIF4E promotes neutrophil survival and accumulation……………………….90 3.2.6 Targeting eIF4E phosphorylation in the tumor and TME prevents metastasis…..97 3.3 Discussion……………………………………………………………………………………………………………………….98 3.4 Materials and Methods ………………………………………………………………………………………………..102 3.5 Acknowledgements…….…………………………………………………………………………………………………107 Chapter 4: General Discussion…………………………………………………………………………………………108 4.1 How does eIF4E phosphorylation affect translation?…………………………………………………….109 4.1.1 Switch to alternative mechanisms of translation initiation……………………………..109 4.1.2 Modulating protein-protein interactions………………………………………………………..111

4.1.3 Promoting initiation factor recycling after 43S PIC recruitment……………………..112 4.2 Revisiting the pro-metastatic effect of eIF4E phosphorylation in cancer cells.……………..115 4.3 Other inhibitors in immune space………………………………………………………………………………….117 Conclusion and perspectives……………………………………………………………………………………………….120 References..…………………………………………………………………………………………………………………….121 Appendix A: supplemental tables and figures………………………………………………………………….153

LIST OF TABLES AND FIGURES: Table 1.1 Relative affinity of eIF4E for various cap analogs…………………………………………………..7 Figure 1.1 Translation initiation………………………………………………………………………………………………5 Figure 1.2 3D structure of cap-bound eIF4E and 4E-BP1 peptide……………………………………………8 Figure 1.3 Cancer inputs and outputs…………………………………………………………………………………….14 Figure 1.4 The translation apparatus plays a pivotal role in mediating the effects of commonly dysregulated oncogenic signaling pathways in cancer………………………………………………………….19 Figure 2.1 eIF4E phosphorylation enhances in vitro metastatic properties…………………………..50 Figure 2.2 eIF4E phosphorylation promotes the translation of mRNAs involved in EMT/invasion and metastasis………………………………………………………………………………………………………………………54 Figure 2.3 Overexpression of eIF4E or phospho-eIF4E targets rescues invasion…………………..56 Figure 2.4 eIF4E is phosphorylated downstream of MAPK during TGFβ-induced EMT………….59 Figure 2.5 eIF4E phosphorylation is required for TGFβ-induced EMT……………………………………63 Figure 2.6 eIF4ES209A mice are resistant to mammary tumor development and metastasis..66 Figure 2.7 eIF4E phosphorylation correlates with expression of SNAIL and MMP-3 in MMTV- PyMT tumors…………………………………………………………………………………………………………………………68 Figure 3.1 Phosphorylated eIF4E in the TME of breast cancer patients correlates with poor survival………………………………………………………………………………………………………………………………….79 Figure 3.2 Phosphorylation of eIF4E in the TME promotes lung metastasis but has no effect on primary tumor growth………………………………………………………………………………………………………….82 Figure 3.3 Neutrophils accumulate in tissues of tumor-bearing WT and eIF4ES209A mice…..86 Figure 3.4 Anti-Ly6G depletion of neutrophils impairs metastasis………………………………………..88 Figure 3.5 Phosphorylation of eIF4E promotes neutrophil survival in response to G-CSF……..91 Figure 3.6 Merestinib inhibits eIF4E phosphorylation, neutrophil survival and metastatic progression of 66cl4 tumors………………………………………………………………………………………………..93 Figure 3.7 MNK inhibition blocks eIF4E phosphorylation, neutrophil survival and metastatic progression of 66cl4 tumors………………………………………………………………………………………………….95 Appendix A: Supplemental tables and figures……………………………………………………………………..153 Supplemental Table 1.1: Abbreviations…………………..………………………………………………….153 Supplemental Table 1.2: eIF4E dysregulation in human cancers………………………………………….154

Figure S2.1. YB1 cannot explain invasion differences…………………………………………………………..155 Figure S2.2. Clustered pathways of eIF4E phosphorylation regulated mRNAs…………………….156 Figure S2.3. Complete list of mRNAs clustered by the DAVID software………………………………158 Figure S2.4. Non phospho-eIF4E regulated mRNAs involved in invasion and metastasis…….160 Figure S2.5. eIF4E phosphorylation does not affect Snail mRNA levels……………………………….161 Figure S2.6. Inhibition of MNKs maintains tight junctions during TGFβ induced EMT………..161 Figure S2.7. eIF4E phosphorylation correlates with EMT and metastatic potential in vivo….162 Figure S3.1. Scoring and histopathology of breast cancer samples………………………………………163 Figure S3.2. Similarities between tumors and immune cells of WT and eIF4ES209A mice…..164 Figure S3.3. T cells and NK cells are unlikely candidates to mediate the effects of eIF4E phosphorylation on the TME……………………………………………………………………………………………….165 Figure S3.4. Neutrophil function and differentiation…………………………………………………………..166

ABSTRACT Most cancer related deaths are due to metastatic progression. While deregulated protein synthesis in cancer cells is recognized as playing a major role in tumorigenesis, the association between the translational machinery and cancer dissemination have remained largely unexplored. To begin addressing this situation, the present thesis focuses on a single regulatory event: the phosphorylation of the cap-binding protein eIF4E. eIF4E is an oncogene that is overexpressed in human cancers, and its levels and activity correlate with poor survival. Phosphorylation on serine 209 is required for its oncogenic activity, and mice bearing a non phosphorylatable alanine mutant of eIF4E (S209A) are resistant to the development prostate cancer. Utilizing these mice and cells isolated therefrom, the current work establishes that cancer cells unable to phosphorylate eIF4E display reduced epithelial- to-mesenchymal transition (EMT) and invasive capacities. Furthermore, mammary tumors bearing the S209A mutation are less metastatic than their wild-type counterparts due to reduced translation of mRNAs encoding MMP3 and SNAIL. Surprisingly, eIF4E phosphorylation in non-transformed cells of the host also plays a role in metastatic progression of mammary tumors, as its detection in the tumor microenvironment correlates with poor survival in breast cancer patients. Consistently, S209A mice are resistant to metastasis, even when bearing tumors derived from 66cl4 cells that express wild-type eIF4E. Neutrophils are, at least in part, responsible for this phenotype: their aberrant accumulation is critical for lung colonization from 66cl4 tumors, however, the translation of anti-apoptotic mRNAs necessary for neutrophils to survive and accumulate is dependent on eIF4E phosphorylation. Interestingly, the small molecule inhibitor merestinib inhibits eIF4E phosphorylation in 66cl4 cells and in neutrophils in vivo, resulting in reduced neutrophil accumulation and the prevention of metastasis. Thus, the experiments outlined herein highlight the importance of translation in the metastatic process, uncover its important role in cells of the tumor microenvironment, and identify a therapeutic target whose inhibition in both the tumor and immune system contributes to the prevention of metastasis.

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RÉSUMÉ La mortalité chez les patients cancéreux est principalement associée à la formation de métastases. Alors que l’importance de la synthèse protéique dans la formation des tumeurs est bien établie, son rôle dans leur dissémination demeure inexploré. Pour étudier cet aspect du cancer, la présente thèse porte sur un phénomène régulatoire important dans la traduction des ARNm en protéine : la phosphorylation d’eIF4E. Ce facteur d’initiation est un oncogène qui est fréquemment surexprimés dans divers cancers, et son hyperactivité est associée à un mauvais pronostic. La phosphorylation sur la serine 209 est requise pour son activité : ainsi, il a été démontré que les souris porteuses d’une mutation à ce site (S209A) sont résistantes au développement du cancer de la prostate. À l’aide de ce modèle animal, cet œuvre permet d’établir que les cellules cancéreuses incapables de phosphoryler eIF4E sont moins aptes à procéder à la transition épithéliale-mésenchymateuse et à envahir la matrice extracellulaire. De plus, les tumeurs mammaires S209A sont moins métastatiques que les tumeurs de type sauvage, ce qui est causé par leur capacité réduite à traduire les ARNm encodant MMP3 et SNAIL. Surprenamment, la phosphorylation d’eIF4E dans les cellules non-transformées de l’hôte joue aussi un rôle dans le processus métastatique, puisqu’il y a une corrélation entre son expression dans les cellules du microenvironnement tumoral et la survie des patientes atteintes du cancer du sein. De plus, les souris S209A sont résistantes à la formation de métastases, même lorsque celles-ci proviennent de tumeurs 66cl4 exprimant la version sauvage d’eIF4E. Les neutrophiles sont responsables de ce phénomène, puisque leur accumulation anormale est requise pour la colonisation des poumons par les cellules 66cl4, alors que la traduction d’ARNm anti-apoptotiques nécessaires à leur survie et leur accumulation dépend de la phosphorylation d’eIF4E. De plus, un inhibiteur de plusieurs kinases, merestinib, bloque la phosphorylation d’eIF4E dans les cellules 66cl4 et les neutrophiles in vivo, permettant de prévenir leur accumulation et la formation de métastases. Ainsi, cette étude souligne l’importance de la traduction dans la dissémination métastatique, a permis de découvrir son rôle dans les cellules du microenvironnement tumoral, et identifie une cible thérapeutique dont l’inhibition à la fois dans la tumeur et le système immunitaire permet de combattre les métastases.

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PREFACE AND CONTRIBUTION OF AUTHORS The present thesis consists in large part of manuscripts published or submitted to peer- reviewed journals, of which I am the first or co-first author. Their citations are included here, with permission where required, and are distributed in the text as follows:

Chapter 1: Robichaud N, Ruggero D, Schneider RJ, Sonenberg S. Translational Control of Cancer. In: Mathews MB, Sonenberg N, & Hershey JWB, editors. Translation Mechanisms & Regulation. Cold Spring Harbor: Perspectives; (expected publication 2018).

Robichaud N, Sonenberg N. eIF4E and Its Binding Proteins. In: Parsyan A, editor. Translation and Its Regulation in Cancer Biology and Medicine. Dordrecht: Springer Netherlands; 2014. p. 73-113.

Bhat M, Robichaud N, Hulea L, Sonenberg N, Pelletier J, Topisirovic I. Targeting the translation machinery in cancer. Nat Rev Drug Discov. 2015 Apr;14(4):261-78.

Robichaud N, Sonenberg N. Translational control and the cancer cell response to stress. Curr Opin Cell Biol. 2017 Apr;45:102-109.

Chapter 2: Robichaud N, del Rincon SV, Huor B, Alain T, Petruccelli LA, Hearnden J, Goncalves C, Grotegut S, Spruck CH, Furic L, Larsson O, Muller WJ, Miller WH, Sonenberg N. Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3. Oncogene. 2015 Apr 16;34(16):2032-42.

Chapter 3: Robichaud N, Hsu BE, Istomine R, Alvarez F, Blagih J, Ma EH, Morales SV, Dai DC, Souleimanova M, Guo Q, del Rincon SV, Miller WH, Jones R, Piccirillo C, Siegel PM, Park M,

Sonenberg N. Translational Control in the Tumor Microenvironment Promotes Lung Metastasis via Phosphorylation of eIF4E in Neutrophils. (submitted to Cancer Cell 2017)

Contribution of authors: The manuscripts forming the major part of this thesis included work from several co-authors to whom I owe many thanks. My contributions and those of my co-authors are delineated here.

Chapter 1 is a literature review consisting mainly of a manuscript entitled “Translational Control of Cancer”, which will constitute a chapter of the upcoming book “Translation Mechanisms & Regulation” for Cold Spring Harbor Perspectives. The original draft and all figures were my work, whereas all authors contributed extensive editorial work on numerous subsequent drafts. Chapter 1 also includes excerpts from other published reviews. For “Translational control and the cancer cell response to stress” and “eIF4E and its binding proteins”, I wrote the original draft and produced the figures, N.S. provided editorial support. For “Targeting the translation machinery in cancer”, I wrote parts of the original draft, revised the manuscript and produced the figures. L.H. wrote most of the first draft. I.T., J.P. and N.S. provided insight, support and revisions. M.B. also participated.

Chapter 2 was published in the journal Oncogene with the title “Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3” Of note, S.V.d.R. and I contributed equally to the work. I contributed little to figures relating to TGFβ- induced EMT, where my only input was to provide reagents (constructs and small molecule inhibitors). I planned, performed and analyzed the experiments presented in all other figures, with guidance from N.S. I drafted the manuscript and compiled the figures, with editorial input from all authors. Some replication experiments were performed by co- authors, and O.L. performed most of the bioinformatics analysis presented in figure 2.2A. S.V.d.R. planned, performed and analyzed experiments on the induction of EMT in vitro, contributed to the writing and editing of the manuscript and the design of the figures. B.H.,

L.A.P., C.G., T.A., and S.G. participated in experiments on EMT. J.H. participated in rescue experiments on invasion. L.F. and W.J.M. were involved in mouse model development. C.H.S., W.H.M. and N.S. secured funding and supervised the research.

Chapter 3 has been submitted to Cancer Cell with the title “Translational Control in the Tumor Microenvironment Promotes Lung Metastasis via Phosphorylation of eIF4E in Neutrophils”. While the list of authors is extensive, the work presented in the main figures and text was planned, performed, analyzed and drafted by N.R., except for the clinical data associated with the tissue microarray, which was collected by M.P.’s group and curated by M.S., and merestinib treatments of 66cl4 cells in vitro, which were performed by S.V.M. and D.C.D. Other contributions from co-authors were included in the supplemental figures or were provided in the form of sample preparation and preliminary experiments. B.E.H., R.I., F.A., J.B. and E.M. were involved in flow cytometry and neutrophil isolation experiments. Q.G. and S.V.d.R. were involved in mouse colony maintenance. W.H.M., R.J., C.P., P.M.S., M.P. and N.S. secured funding, recommended experiments, supervised the research and edited the manuscript.

Contributions to knowledge and elements of original scholarship: This document has been eight years in the making. It is therefore not surprising that elements that were considered original at the time have since been described by other groups as well. This fact may lead to some unfortunate confusion when reading the literature review introducing my thesis, as it reviews work published as recently as August 2017. It therefore cites publications with experiments that would seem to take away from the novelty of my own investigations, but is merely a consequence of their release at a time near, or subsequent to, that of my own data. To avoid any misunderstandings, the elements of this thesis which were considered original scholarship at the time the research was conducted are delineated below. A more explicit description of publications preceding and following the experiments described here are included as the introduction of each chapter.

Elements that are not as original but nonetheless constitute distinct contributions to knowledge are also described here.

Elements of original scholarship:

1. Phosphorylation of eIF4E promotes migration and invasion in several cell models. 2. eIF4E phosphorylation promotes the translation of the mRNA encoding SNAIL. 3. eIF4E is phosphorylated downstream of TGFβ 4. Phosphorylation of eIF4E promotes EMT independently of the activity of the SMAD family of transcription factors. 5. Phospho-eIF4E promotes mammary tumor formation in the MMTV-PyMT mouse model 6. eIF4E phosphorylation promotes metastatic progression in vivo. 7. Phospho-eIF4E is detectable in the stromal cells of human breast cancer samples, and its presence correlates with poor survival. 8. Translational control in the TME plays a significant role in tumor biology 9. Phospho-eIF4E in the TME promotes metastatic progression 10. Phospho-eIF4E promotes the survival of neutrophils in response to G-CSF 11. Merestinib inhibits metastasis in a mouse mammary tumor model

Distinct contributions to knowledge Other aspects were not entirely original but served to reinforce or dispute previously published observations:

1. The importance of translational control in cancer is well established; the current work contributes to the broader acceptance of this concept, in particular as it pertains to the phosphorylation of eIF4E. 2. Many of the known activities of eIF4E have been shown to require its phosphorylation for maximal effect, notably cellular transformation and cytokine production. Now, EMT, invasion and metastasis can be included in this list.

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3. Activation of the MNKs has been linked to EMT in glioblastoma, but the mechanism invoked differed entirely and involved SMAD2, which we tested but could not observe. 4. Translation in cells of the TME has been largely ignored, despite reports indirectly supporting its potential role. Thus, eIF4F complex formation in vitro has been suggested to be affected by crosstalk between multiple myeloma cells and mesenchymal stem cells, and mTOR activity is important in both myeloid-derived suppressor cells and T cells in several disease models. The experiments herein explicitly establish a role for translation in the TME and demonstrate its effect on metastatic progression, solidifying previous findings and providing a solid rational for future investigation of this concept.

ACKNOWLEDGEMENTS First and foremost, I would like to thank Nahum for the incredible freedom and trust he has given me throughout my PhD. None of this work would have been possible without his support and guidance. I am extremely thankful for the extraordinary learning opportunities that he has provided me, which are beyond the normal scope of graduate work. Second, I would like to thank a few other important mentors that were always available for interesting discussions, advice, and helped shape my scientific mind: Luc Furic, Ivan Topisirovic, Nadeem Siddiqui, and the regretted Colin Lister. Third, there are several professors that generously offered their time and advice, so thank you to (in alphabetical order) Drs Connie Krawczyk, Wilson Miller, William Muller, Robert Nadon, Morag Park, Arnim Pause, Jerry Pelletier, Ciriaco Piccirillo, Peter Siegel, Michel Tremblay, Josie Ursini-Siegel, and Ian Watson.

I am also grateful for the financial support I have received in the form of grants and scholarships from government agencies, CIHR and FRQ-S, as well as private foundations, Susan G. Komen for the Cure and Terry Fox Research Institute. Even more so for the amazing fundraising in the Montreal community, led by the Goodman family and the wonderful people at Canderel, that allow for the creation of scholarships and travel awards that have been so important for my PhD research and that of my peers. Therefore, I extend my thanks

xiii to the donors, organizers and armies of students and staff members involved in these efforts.

I would also like to acknowledge everyone that was involved in the experiments described herein. This includes all my co-authors in manuscripts included in this thesis, whose contributions are detailed in the previous section, as well as several other people who taught me how to perform critical experiments. I would therefore like to thank Chadi Zakaria for taking over the midi-prep I for some reason could never get to work, and for putting up with my sense of humor and general shenanigans; Colin Ratcliffe, Dongmei Zuo and the good people at the imaging facility for their help, training and advice on microscopy; Nadeem Siddiqui and Edna Matta Camacho for their advice on recombinant protein production and assays using them (too bad that project never led to anything…); Isabelle Harvey and Meena Vipparti for all the work they do for everyone in the lab; Annie Sylvestre, Annik Lafrance and everyone at McGill’s Comparative Medicine and Animal Resources Centre, for all their help and training on experiments using mice; Julien Leconte and Vinicius Motta for flow cytometry training and cell sorting; and everyone at the histology facility for processing of mouse samples for immunohistochemistry. Many thanks to the following people for editorial help on various published works: Valerie Henderson, Dana Pearl, Soroush Tahmasebi, Tommy Alain and Chadi Zakaria. If I’ve forgotten anyone, please forgive me and accept these anonymous acknowledgements instead: Thank you everyone I’ve worked with over the last 8 years.

Finally, I would like to thank my family for all the support I’ve been lucky to have. My wife, Jida, for constant motivation and for just being awesome at everything. My daughter Naïad, for being a permanent joy in my life. And, also: happy hour.

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CHAPTER 1

GENERAL INTRODUCTION

RATIONAL AND OBJECTIVES Translational control plays a critical role in the regulation of gene expression, and its role in tumorigenesis is well established. Among other effects, deregulated translation in cancer promotes the unchecked proliferation, enhanced survival and aberrant vascularization associated with the development of primary tumors. However, in many cancer types including breast cancer, primary tumors are not lethal; rather, the eventual metastatic dissemination of cancer cells is responsible for disease-related mortality. Yet the importance of translation in this process has remained largely unexplored. Furthermore, many non- transformed cell types are of critical importance to metastasis. While these cells must translate mRNAs into proteins for their function, there is a severe dearth of information on how they regulate translation in the cancer setting. These gaps in our knowledge constitute an important problem to address, as there is a long list of inhibitors of translation in clinical trials, yet we know almost nothing regarding how they will affect metastasis, or the function of the immune system. The work presented in this thesis is based on the hypothesis that translation plays an important, as of yet unappreciated, role in the metastatic process by regulating the ability of cancer cells to disseminate and the ability of immune cells to respond to cancer. As a first step in correcting this deficiency, the work presented in this thesis proposes to focus on a single regulatory event for which we possess an ideal mouse model, the phosphorylation of the eukaryotic initiation factor 4E, in order to achieve the following objectives:

1. Investigate the role of translation in the metastatic dissemination of cancer cells; 2. Investigate the role of translation in cells of the tumor microenvironment; 3. Determine the impact of targeting translation in both the tumor and host.

To fully appreciate the significance of this endeavor, one must be familiar with the current state of knowledge in the field of translational control of cancer. Therefore, a review of the relevant literature is presented here.

1. LITERATURE REVIEW 1.1 On the importance of translational control in cancer 1896: the modern Olympics were created, Wilhelm Röntgen described X-rays for the first time, Henri Becquerel discovered radioactivity and Giuseppe Pianese associated nucleolus size with cancer malignancy. While Pianese’s discovery never reached the kind of fame and impact associated with these other achievements, it was the first indication, only to be understood a century late, that the translation of mRNAs to proteins is critical for cancer development and progression. Of course, Pianese could not have known the mechanism underlying the association between nucleoli and cancer in the 19th century, or even the term “nucleolus”. Nonetheless, the importance of his discovery is demonstrated by the fact that today, in the modern era of molecular pathology, the number and size of nucleoli are still used as prognostic biomarkers for clinical outcome and tumor recurrence (1).

We now know that the nucleolus is the site of ribosome biogenesis, and cancer cells display larger nucleoli to support the increased ribosomal biosynthesis required for hyperactive proliferation. In fact, increased ribosome content is critical during cell cycle progression to ensure adequate doubling of protein content that signals cell division. Ribosomes themselves are enormously abundant, must be duplicated during cell growth to promote proliferation, and are thought to coordinate the two processes. One hypothesis proposes that mRNAs encoding ribosomal proteins are so abundant that they act as sponges, sequestering ribosomes from the low-abundance mRNAs necessary for cell cycle progression, until sufficient mature ribosomes are available for the translation of other transcripts (2).

While this hypothesis has a certain elegant appeal, other mechanisms are also known to be at play, which are the subject of some excellent reviews (3, 4). Nonetheless, the abundance of ribosomes serves to remind us the extent of the resources dedicated to support mRNA translation. Indeed, in proliferating cells, up to 20% of cellular energy is used for translation, compared to 15% for transcription and replication, and 20% for various cation pumps (5). As most of transcription is directed to the synthesis of ribosomal RNA (rRNA) and mRNAs encoding

3 ribosomal proteins, it is clear that translation is easily the most energy demanding cellular process, which has been corroborated in vivo (6).

Considering the expense of dedicating so much energy to mRNA translation, this crucial step in gene expression is heavily regulated and has a profound impact on variations in protein levels. There have been several attempts to dissect the relative contributions of gene transcription, mRNA stability, mRNA translation and protein degradation on the steady-state levels of cellular proteins (7-10). These studies have highlighted the important differences between the transcriptome and proteome, with variations in mRNA levels accounting for 30-80% of variations in protein levels in cells and tissues. This moderate correlation is mostly attributed to translational regulation, as there is more than a 1000 fold difference in the translation rates of various mRNAs (7, 10). It is worth noting that there is some concern that experimental caveats may have affected the conclusions reached from some of these studies (11). However, the overwhelming consensus is that translation plays a major role in regulating gene expression, particularly in specific conditions such as cellular stress and cell growth (9, 12), two very important aspects of tumor biology.

1.2 Brief overview of translational control Translation occurs in three steps: initiation, which includes all the steps leading up the recognition of the start codon; elongation, consisting of the sequential addition of amino acids corresponding to the mRNA sequence; and termination at the stop codon allowing ribosomes to be released or enter further rounds of translation. Our current understanding is that the key regulatory events are the formation of two trimeric complexes: eIF4F and the Ternary Complex (TC) (Figure 1.1). The TC’s monopoly of the initiator methionine tRNA (Met-tRNAi) ensures that this complex is crucial for start codon recognition and translation initiation. eIF4F is responsible for mRNA recruitment and is required for the unwinding of secondary structures present in the 5’UTR of most cellular mRNAs (13).

Figure 1.1. Translation initiation. The rate-limiting phases of initiation involve controlling the assembly of a functional 40S subunit with its associated factors (43S pre-initiation complex (43S PIC)) and the access of PICs to the mRNA template (14, 15). The 43S PIC is a large multifactorial complex formed by the association of the 40S ribosomal subunit with eukaryotic translation initiation factors (eIFs) eIF1, eIF1A, eIF3, eIF5 and the ternary complex (TC) (16, 17). The TC consists of a trimeric complex involving eIF2 (containing α-, β- and γ-subunits), initiator methionyl tRNA (tRNAiMet) and GTP (18). The recruitment of the 43S PIC to the mRNA template is facilitated by eIF4F, a complex consisting of the mRNA 5′-cap-binding subunit (eIF4E), a large scaffolding protein (eIF4G) and the DEAD box RNA helicase (eIF4A) (19, 20). eIF4F recruits ribosomes to mRNA through eIF4E–mRNA cap and eIF4G–eIF3 interactions, resulting in the formation of a 48S initiation complex (21). eIF4G also interacts with the poly(A)- binding protein (PABP), which associates with the mRNA 3′ poly(A) tail, to cause mRNA circularization to stabilize mRNAs and bolster translation (22). The eIF4A helicase participates in the initial interactions of eIF4F with the mRNA 5′ end and may also facilitate scanning of the 40S ribosomal subunit towards the initiation codon by resolving the secondary structure in the 5′ untranslated region (UTR) (23). Recognition of the initiation codon by the 43S PIC leads to the release of eIFs and joining of the 60S ribosomal subunit (23). The formation of a translation- competent 80S ribosome marks the end of initiation and the beginning of elongation. BCL-XL, B- cell lymphoma extra large; 4E-BP, eIF4E-binding protein; m7G, 7-methylguanosine 5′-cap; MNK, MAPK-interacting kinase; VEGF, vascular endothelial growth factor.

In addition to these complexes, other initiation factors play important roles. These include eIF3, the bridge between eIF4F and the pre-initiation complex (PIC) (24); eIF5A, which appears to be involved in initiation, elongation, as well as other steps in mRNA metabolism (25); and eIF6, which is involved in ribosome biogenesis in the nucleolus and prevents premature joining of the 40S and 60S ribosomal subunits in the cytoplasm (26). As this thesis revolves around eIF4E and its phosphorylation, these topics are expanded upon here.

1.3 eIF4E Binds the Cap Nuclear transcribed mRNAs possess a cap structure (m7GpppN, where m7G is 7-methylguanosine and N is any nucleotide) at their 5’ end, which is critical for mRNA splicing and polyadenylation, stability and translation. eIF4E was first discovered as a 24 kDa protein that could be chemically crosslinked to the cap (27, 28). The structure of eIF4E resembles that of a cupped hand pinching the cap between finger and thumb (29-31). The molecular basis for the specificity of eIF4E for capped mRNAs is described in Figure 1.2. The most important interaction is the stacking of the guanine ring between two aromatic tryptophans, which is strengthened by delocalized positive charge arising from the methyl group of the cap structure. Further interactions occur between the cap-binding pocket and the guanine, the methyl group, the triphosphate bridge and the second nucleotide (Figure 1.2A-D). Each of these interactions is important for cap recognition, as demonstrated by the hierarchy of eIF4E’s affinity to various cap analogs (32, 33) (summarized in Table 1.1). Impairing eIF4E’s cap-binding activity by mutating W56 abrogates its oncogenic effects in mouse models and human cell lines (28).

Table 1.1 Relative Affinity of eIF4E for Various Cap Analogs

Interaction Relative binding Second nucleotide m7GpppA>m7Gppp Triphosphate bridge m7GpppA>m7GppA m7Gppp>m7Gpp>>>m7Gp Methyl group m7Gppp>>>Gppp m7: methyl group on position N7 of the guanine; G: guanosine; A: adenosine; p: phosphate group Ref: (32, 33)

Figure 1.2. 3D structure of cap-bound eIF4E and 4E-BP1 peptide. A, aromatic stacking of the purine ring between Trp 56 and Trp 102 and electrostatic interactions between N1/N2 and Glu 103; B, van der Waals interactions between the methyl group and Trp 166; C, electrostatic interactions of the triphosphate bridge with Arg 157 and Lys 162; D, interactions between the second nucleotide and residues of the flexible C-terminal loop of eIF4E. Structure from PDB 1WKW (30).

1.4 Regulation of eIF4E 1.4.1 eIF4E interacting proteins: eIF4G The first protein that was described as a binding partner of eIF4E was eIF4G (20, 34, 35). The human genome encodes two functionally redundant eIF4G family members: eIF4GI and eIF4GII (36). These proteins bind to eIF4E, eIF3 and eIF4A, and can restore cap-dependent translation in rabbit reticulocyte lysates treated with rhinovirus 2Apro (20, 36). A third family member, DAP5, cannot bind eIF4E and is involved in cap-independent translation. eIF4GI and II are ubiquitously expressed, though their relative expression varies in different tissues (36). eIF4GI was the first identified homolog and is generally used in studies on eIF4E-eIF4G interactions. Therefore, it is referred to as eIF4G in this thesis and the numbering of important residues relate to this homolog. Two conserved residues in eIF4E are critical for its interaction with eIF4G, Val69 and Trp73 (37). eIF4G interacts with eIF4E via the sequence YDREFLL, which conforms to the canonical eIF4E-binding motif, YXXXXLφ (where X is any amino acid and φ is hydrophobic). RNA binding motifs in mammalian eIF4G stabilize the interaction of the eIF4F complex with the cap by maintaining eIF4E in the vicinity of the cap, increasing its local concentration and favoring the bound state (38). The RNA binding motifs on eIF4G likely do not affect the on/off binding rates of eIF4E to the cap structure itself (39).

1.4.2 eIF4E interacting proteins: 4E-BPs The 4E-BPs are small (~15-20 kDa) proteins that interact with eIF4E (40). There are three known isoforms in mammals (4E-BP 1, 2, 3). Relatively little is known regarding 4E-BP3, especially in cancer. Most studies focus on 4E-BP1, and to a lesser extent 4E-BP2. Both isoforms 1 and 2 are ubiquitously expressed, although the predominant species varies across different tissues (41, 42). As these isoforms are functionally redundant in the context of cell growth and cancer (43), they are jointly referred to as the 4E-BPs. The 4E-BPs are regulated by the mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates several residues in a hierarchical manner. First, Thr37/Thr46 are phosphorylated by the mTOR kinase, followed by Thr70 and finally Ser65 (44). Thr70 and Ser65 are responsive to extracellular cues such as serum

10 stimulation (44). Phosphorylation of all of these sites (the hyper-phosphorylated form) inhibits 4E-BPs’ binding to eIF4E. In this manner, mTORC1 regulates eIF4E and translation.

The 4E-BPs act as inhibitors of eIF4E function by competing with eIF4G for binding to the dorsal side of eIF4E (45, 46), preventing the formation of the eIF4F complex for subsequent translation initiation (40). eIF4G and the 4E-BPs bind to eIF4E via their conserved eIF4E-binding motif (YXXXXLφ) using a similar disorder-to-order transition mechanism, and possess similar affinities for eIF4E (37, 47-49). Despite this, the kinetics of binding differ widely, as the 4E-BPs display rates of binding and dissociation two to three orders of magnitude faster (50). Conceptually, this difference is consistent with the inhibitory role of the 4E-BPs, requiring rapid control of eIF4E binding, whereas eIF4G requires longer-lived interactions with eIF4E to promote translation initiation. Recent studies have discovered the molecular basis for this important distinction by uncovering a second eIF4E-binding site that differs between eIF4G and the 4E-BPs (50, 51). A structural study using full-length 4E-BP2 has confirmed the importance of the second binding site: while 4E-BP2 is intrinsically disordered, both eIF4E-binding sites possess significant transient secondary structure and contribute to 4E-BP2’s affinity for eIF4E (49). The dynamic nature of the eIF4E/4E-BPs interaction is attributed to the second binding site (49).

1.4.3 eIF4E phosphorylation eIF4E is phosphorylated on a single site, Ser209, by the map kinase integrating kinases (MNK1/2), which are activated in response to cellular stress and survival signals from the MEK/ERK and p38 MAPK pathways (52, 53). Phosphorylation requires prior binding of the MNKs to eIF4G, indicating that phosphorylation of eIF4E occurs after the formation of the eIF4F complex (54). eIF4E phosphorylation is not required for global translation as mutation of Ser209 to alanine or genetic abrogation of the MNKs has no deleterious effects in in vivo models (55, 56). Rather, eIF4E phosphorylation controls the translation of a specific subset of mRNAs, although the mechanism by which this occurs is uncertain. Originally, phosphorylation on S209 was predicted to increase eIF4E’s affinity for the cap by establishing a salt bridge with lysine 159, clamping down on bound mRNAs (29, 57); however, the distance between the two

11 residues appears to be too great for such an interaction (58). Biophysical studies including surface plasmon resonance, stopped-flow kinetics and fluorescence titration experiments indicate that eIF4E phosphorylation decreases the affinity for the cap (59-61). This is due to electrostatic repulsion between the phosphorylated S209 and the intrinsic negative charges within the cap, a hypothesis supported by the fact that increasing the number of phosphate groups present in cap analogs exacerbates the effect of eIF4E phosphorylation (61). Phosphorylation by the MNKs requires eIF4G, and thus occurs after eIF4F complex formation (54). It has also been reported to require eIF3, suggesting that phosphorylation occurs after PIC recruitment (62). Thus, eIF4E phosphorylation promotes dissociation of eIF4E from the cap once scanning of the 5’UTR has begun. This may facilitate the dissociation of eIF4E from the mRNA and its recycling for further rounds of translation (63). Rapid recycling of eIF4E would be approximately equivalent to punctual increases in available eIF4E, which may explain why 5’UTR elements conferring sensitivity to eIF4E levels also confer sensitivity to eIF4E phosphorylation (64), and why many of the identified targets responding to eIF4E phosphorylation are targets of total eIF4E. However, this hypothesis is still lacking direct evidence.

1.5 Deregulated translation in cancer Aberrant expression and phosphorylation of eIF4E have been linked to cancer, as have the other components of the eIF4F complex, regulators of TC formation, and many other initiation factors, as listed in Supplemental Table 1.2 (Annexe A). How do cancer cells highjack the translation machinery for their growth, survival and expansion to form tumors? And importantly, how do these changes in the activity of distinct translation factors, assumed to be important for the translation of all cellular mRNAs, confer specificity to the translation of selective mRNAs?

1.5.1 Specific effects of general translation initiation factors Curiously, the mechanism underlying the mRNA-specific effect of changes in general initiation factors was worked out before many of the factors involved were even identified. Indeed, a

12 mathematical model was derived demonstrating that, given constant elongation rates and limited PIC concentrations, mRNAs for which ribosome recruitment is inefficient will be disproportionately affected by any changes in translation initiation capacities (65). While based on rudimentary knowledge of ribosomal function, this model was sufficient to predict the response of α- and β-globin synthesis to inhibitors of translation. It can even be used to explain translational deregulation in cancer cells with our current understanding of this process, if we assume elongation rates are approximately constant and that PIC concentrations are low enough to allow for this disproportionate effect to occur, possibly due to oncogenic stress signals leading to eIF2α phosphorylation. In this context, any strategy taken by cancer cells to favour general ribosomal subunit joining at start codons should disproportionately improve the translation of mRNAs with poor initiation efficiencies. This is crucial for cancer cells, as it turns out that transcripts encoding oncoproteins tend to be poorly translated, purportedly to maintain tight control of their expression in normal cells, thus preventing transformation. Examples of such transcripts include those encoding the vascular endothelial growth factor VEGFA, the anti-apoptotic proteins BCL2 and MCL1, the oncogene MYC, as well as several cyclins and cyclin-dependent kinases. Thus, the model predicts that cancer cells should be particularly well equipped to maintain angiogenesis, survival and proliferation, even in times of stress, simply by increasing the availability of general initiation factors, or by removing the brakes on particularly poorly translated mRNAs. Some of the many ways by which this can be achieved are delineated here and summarized in Figure 1.3.

1.5.2 Mechanisms of deregulated translation in cancer (cancer inputs) 1.5.2.1 Changes in cis: 5’ and 3’UTR length and composition Sequence and structural motifs present in mRNAs determine their intrinsic translational efficiency and their ability to be regulated by trans-acting factors such as miRNAs, RNA-binding proteins and initiation factors. These motifs tend to be overrepresented in oncogenic transcripts, thus rendering these transcripts intrinsically poorly translated and subject to the kind of disproportionate regulation proposed by Lodish in 1974.

Figure 1.3: Cancer inputs and outputs. Summarized view of the oncogenic lesions feeding into the translational machinery (cancer inputs) and of the resulting advantages conferred by aberrant translation, with examples of regulated mRNAs (cancer outputs).

Increased secondary structure (stability) of the 5’ untranslated region (5’UTR) was one of the first cis-acting elements to be identified that affect the rate or efficiency of cap-dependent translation initiation. This was demonstrated by artificially enhancing base-pairing in reovirus mRNA by substitution of uridines for bromouridines, leading to increased dependence on eIF4E for translation; inosine substitution had the converse effect on secondary structure and translation efficiency (66). Oncogenic transcripts possessing stable 5’UTRs, and thus dependent on eIF4E, include those encoding MYC, ODC, and several cyclins and cyclin dependent kinases. Many other mRNA sequence elements can regulate the efficiency of translation. For example, exquisite dependence of eIF4E, but not eIF4A, has been demonstrated for the Translation Initiator of Short 5′UTR (TISU) element (67), whereas mRNAs containing internal ribosome entry sites (IRESs) are cap- and eIF4E-independent, but dependent on eIF4G and eIF4A (68). IRES- mediated translation allows for direct recruitment of ribosomes to start codons without the requirement for 5’UTR scanning (68). This mechanism is particularly important when scanning is impaired due to limiting eIF4E, such as in stress conditions (68). Furthermore, alternative initiation codons and inhibitory open reading frames upstream of the canonical AUG can severely hamper its identification by the 43S PIC; however, strategic placement of uORFs can lead to selectively increased translation in conditions of eIF2α phosphorylation (13). Additionally, structural or sequence motifs can recruit RNA-binding proteins to modulate mRNA translation, such as the transforming growth factor‑β (TGFβ)-activated translation (BAT) element, an asymmetrical bulge-containing stem-loop that regulates the translation of mRNAs involved in epithelial-to-mesenchymal transition (69). Finally, binding sites for miRNAs are particularly common motifs that affect translation, in addition to mRNA stability, as are AU-rich motifs. All of these elements have been thoroughly reviewed elsewhere (13, 68, 70-72).

A particularly interesting example relates to terminal oligopyrimidine (TOP) motifs, consisting of a C and a series of 4-15 pyrimidines immediately following the cap structure. TOP motifs provide a unique “all-or-nothing” translational regulation to mRNAs encoding components of the translational machinery, though the underlying mechanism remains elusive (73). This was sequentially attributed to phosphorylation of RPS6 by the S6 kinases (74) and to the activity of

16 eIF4E downstream of 4E-BP phosphorylation (75, 76). However, cells lacking S6Ks or 4E-BPs retain the ability to modulate TOP mRNA translation downstream of mTOR (73, 77, 78). Many new candidates have emerged as possible TOP regulators, such as miR-10a/miR-10b, and several RNA-binding proteins: TIA-1/TIAR, ZNF9, La protein and the La-related proteins LARP1 and LARP7 (79-84). LARP1 in particular has garnered much interest, and has been proposed to regulate TOP mRNA stability, to bind to PABP, to bind to the cap, and to either increase or decrease TOP mRNA translation (83, 85-87). An interesting recent finding proposes that LARP1 interacts with the 40S ribosomal subunit to stabilize TOP mRNAs and involves this factor in the pathogenesis of ribosomaopathies (88). Further research into this field will hopefully resolve the controversy and confusion.

The majority of identified cis-acting elements described here reduce mRNA translation efficiency, and can be combined in individual mRNAs for efficient suppression. Since such combinations are commonly observed in oncogenic mRNAs to avoid abnormal expression and transformation, cancer cells can evolve a variety of mechanisms to bypass this control. The simplest involve shortening 5’ and/or 3’UTRs to eliminate suppressive elements. For example, cancer cells can use alternative transcription start sites downstream of a regulatory element. This has been observed for the transcription of Mdm2 which can skip two inhibitory uORFs (89). Similarly, inhibitory elements can be avoided by using alternative polyadenylation, leading to shorter 3’UTRs lacking miRNA binding sites, AU-rich elements, and other regulatory elements (90). These mechanisms appear to be wide-spread in cancer cells, whose transcriptomes are globally shorter, thus providing net advantages by removing the brakes on oncogenic mRNAs (91).

1.5.2.2 Oncogenic signaling The most common mechanism for deregulated translation in cancer is probably through the activation of upstream signaling pathways. Indeed, the translational machinery is a major regulatory hub integrating signals from oncogenic signaling cascades to regulate the translation of important target mRNAs. mTOR in particular plays a crucial role by phosphorylating the 4E-

BPs to allow eIF4F complex formation. S6K is also important, as it phosphorylates several substrates such as programmed cell death 4 (PDCD4) to release eIF4A, eEF2K to alleviate inhibition of elongation through eEF2, as well as eIF4B to promote the helicase activity of eIF4F (92-94). In fact, signals from mTOR, MAPK and WNT all funnel to the activation of translation, which has been shown to be required for the transforming potential of these pathways (95). Many of the most commonly mutated genes across cancer types encode proteins in these pathways, including PIK3CA, KRAS, PTEN, APC and EGFR (96). Other commonly mutated pathways, such as NOTCH and SMAD, do not themselves regulate translation but require its concurrent activation to promote the expression of their target genes (95, 97). Particularly interesting is the case of the MYC oncogene, which promotes almost all aspects of translation (see below). Many of these signaling pathways present complex patterns of crosstalk and rewiring in cancer cells, as has been well documented between the mTOR and MAPK pathways (142) and been recently described for mTOR and GCN2 (143). The eventual connection at the translational machinery is a crucial aspect of this circuitry and presents an interesting opportunity for therapeutic targeting, as described at length elsewhere (144). Certainly, translation is far from the only effector of these pathways, yet its importance is demonstrated by the requirement for its activity in the transformed phenotype that they induce, as well as in the speed at which it is modulated (115). For example, following inhibition of upstream kinases such as AKT and KRAS, decreased translation efficiency occurs prior to any transcriptional changes in models of glioblastoma (145). Furthermore, direct deregulation of the translation machinery can occur independently of upstream signaling, or arise as a resistance mechanism to its inhibition, underscoring its significance in cancer (146-151).

1.5.2.3 Gain/loss of initiation factors Aberrant expression of eIFs was the first mechanism identified that allows cancer cells to deregulate translation to their benefit, when eIF4E was found to transform NIH 3T3 cells in vitro (98) and to promote the development of tumors in transgenic mice (99). eIF4E was subsequently found to be overexpressed in a wide variety of human cancers, as were several other initiation factors, all of which have been comprehensively tabulated elsewhere (100-102).

Figure 1.4. The translation apparatus plays a pivotal role in mediating the effects of commonly dysregulated oncogenic signaling pathways in cancer. The signaling cascades by which hyperactive phosphoinositide 3-kinase (PI3K)–mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways induce formation of the eukaryotic translation initiation factor 4F (eIF4F) complex are indicated. Proteins encoded by commonly mutated oncogenes and tumour suppressors in these pathways are shown in yellow and red, respectively. On the left are the stresses leading to eIF2α phosphorylation, which inhibits translation by stabilizing the interaction between eIF2B and GDP–eIF2 (Ref. 235). Also shown is the transcriptional regulation of the translation machinery by the oncoprotein MYC that activates eIF4E, eIF4A and eIF4G and bolsters tRNAs and rRNA synthesis286, whereas inactivation of the p53 and pRB tumour suppressors releases their repression of tRNA and rRNA synthesis287. The net outcome of these alterations differs among tumour cells but is invariably linked to a disproportionate increase in the translation of eIF4E-sensitive transcripts such as MYC. Bar-headed lines indicate inhibition, and arrows indicate activation. The thick dashed arrow indicates nuclear translocation of MYC. Components of the translation machinery leading to neoplastic transformation when ectopically overexpressed are circled with red shading. 4E-BP, 4E-binding protein; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; GCN2, general control non-derepressible 2; HER2, human epidermal growth factor receptor 2; HRI, haem-regulated inhibitor; m7G, 7- methylguanosine 5′-cap; MAPKKK, mitogen-activated protein kinase kinase kinase; MEK, MAPK/ERK kinase (also known as MAPKK); MET, mesenchymal epithelial transition factor; MNK, MAPK-interacting kinase; mTORC, mTOR complex; PABP, poly(A)-binding protein; PDCD4, programmed cell death 4; PDK1, 3-phosphoinositide-dependent protein kinase 1; PERK, PKR-like ER kinase; PIC, pre-initiation complex; PIP2, phosphatidylinositol-4,5- bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKR, double-stranded RNA- dependent protein kinase; RHEB, RAS homologue enriched in brain; PTEN, phosphatase and tensin homologue; S6K, ribosomal S6 kinase; tRNAiMet, initiator methionyl tRNA; TSC, tuberous sclerosis.

1.5.2.3.1 eIF4F complex formation Ribosomes are recruited to the 5’ ends of mRNAs via the eIF4F complex, which consists of eIF4E, eIF4G and eIF4A (Figure 1.1). All three subunits can be deregulated in cancer cells: their genomic loci have all been shown to be amplified in human tumors, and they are all targets of the MYC oncoprotein (see below) (100-102). Of note, the relative importance of eIF4A expression levels is questionable. Of the two homologs with 90% identity that exist in humans, eIF4A1 is the most widely expressed and best studied, but genetic lesions in the Eif4a1 gene are rare in cancer; in contrast, Eif4a2 is amplified in 45% of lung squamous cell carcinomas, though this may be a side-effect of its location on a frequently amplified region of chromosome 3q (103). Indeed, eIF4A is expressed at much higher levels than other initiation factors, and is thus unlikely to be limiting, even in the cancer setting (104). Nonetheless, eIF4A activity is crucial for the translation of mRNAs with stable 5’UTR secondary structures, such as those encoding anti- apoptotic factors (105), and its inhibition leads to potent tumor suppression (106). In contrast, eIF4E and eIF4G act as classical oncogenes, their overexpression resulting in transformation in vitro and in vivo (98, 99, 107, 108). Importantly, it was recently demonstrated that eIF4E expression is normally maintained in excess for cap-dependent translation, and that loss of eIF4E by 50% is not limiting for general protein synthesis and embryonic development. Indeed, eIF4E+/- mice are viable and do not display any obvious phenotypes. On the contrary, eIF4E+/- cells and mice are remarkably resistant to cellular transformation, even when driven by a powerful oncogene such as Hras-V12 (109).

As eIF4E has been the best studied initiation factor in cancer, other modes of deregulation have also been reported, for which similar mechanisms could potentially affect eIF4A and eIF4G. Thus, deregulated eIF4E can be achieved by activation of transcription factors other than MYC, by mRNA stabilization via loss of HuR or Eif4e-targeting miRNAs, as well as by increased phosphorylation (110-112). eIF4E phosphorylation by the MNKs promotes tumor development and dissemination (55, 97, 113, 114), and is elevated in human lung, breast and prostate cancers, among others (115-117). While the MNKs do not appear to be frequently amplified or

21 mutated, their expression can be increased by YB1 (118), though activation of upstream MAPK signaling is likely the most common means of promoting eIF4E phosphorylation in cancer cells.

Alternatively, eIF4F complex formation can be promoted by loss or inactivation of proteins responsible for the sequestration of eIF4A or eIF4E. Thus, despite the uncertain role of eIF4A amplification in cancer, loss of its inhibitor, PDCD4, appears to provide a significant advantage, as it acts as a tumor suppressor whose absence is associated with cancer cell invasion and poor survival in some cancers (119-123). Decreased PDCD4 expression is linked to posttranscriptional regulation by miR-21 and degradation following phosphorylation by S6Ks (93). While it is tempting to attribute PDCD4’s tumor-suppressive properties to eIF4A inhibition, PDCD4 appears to be a multifunctional protein that has also been reported to regulate AKT and JNK signaling, and the transcription factors NF-κB, c-JUN and AP-1, through mechanisms that remain unclear (124-126). As one report attributes most of these functions to downstream effects of MYC regulation (127), the exact role of PDCD4 in cancer remains to be established.

In contrast, the eIF4E-binding proteins (4E-BPs), which compete with eIF4G for binding to eIF4E, clearly act as tumor suppressors via inhibition of cap-dependent translation. The 4E-BPs can either be lost, as occurs in pancreatic cancer but rarely in other cancer types, or functionally impaired by inhibitory phosphorylation via mTOR. Consequently, any activation of the mTOR pathway results in increased eIF4E availability for translation. While several studies linked elevated 4E-BP1 expression to cancer progression, suggesting a role as an oncogene rather than a tumor suppressor (128, 129), it appears that even when overexpressed in the metastatic setting, 4E-BPs are mostly hyper-phosphorylated in human cancers and therefore inactive, negating their overexpression as a cause of transformation. Confirming these data, our own unpublished observations indicate that genetic deletion of 4E-BP1/2 has no impact on tumor progression in a mouse model of PTEN-loss driven prostate cancer, as mTOR is highly active in this model. Of note, it is impossible to completely exclude the possibility that 4E-BP overexpression plays other, non-translational roles in cancer, which has been previously reported (130).

1.5.2.3.2 Pre-initiation complex formation As delineated in Figure 1.1, translation begins with the formation of the 43S PIC formation, which is controlled by phosphorylation of eIF2α. Deregulated PIC formation in cancer cells is a complex issue that has led to some seemingly contradictory (131). On the one hand, it is generally thought that increased eIF2α phosphorylation would grant cancer cells a heightened ability to respond to the various stress conditions encountered along the path to malignancy by promoting the translation of uORF-containing stress-response transcripts such as ATF4 (132). Thus, overexpression of eIF2α or its kinase PKR have been shown to promote transformation in some contexts (133-137). On the other hand, long-term eIF2α phosphorylation engenders apoptosis, and has even prompted research into the development of cancer therapies promoting the activity of eIF2α kinases or the inhibition of their phosphatases (138-140). These results suggest that the outcome of eIF2α phosphorylation in cancer cells is highly context specific, perhaps related to disease site, tumor grade or underlying driver mutations. Likely, the ability of cancer cells to reap the benefits of eIF2α phosphorylation while avoiding its eventual anti-proliferative and apoptotic consequences relies on decoupling of the two processes. This could occur at the molecular level by enhancing translation dependent on alternative mechanisms such as IRESs, mRNA methylation and eIF2A (132, 141-143). Alternatively, chronology may be important, with sequential phosphorylation and quiescence in times of severe stress (e.g. hypoxia at early disease stages), followed by dephosphorylation and proliferation after adaptation (e.g. neo-vascularization) (101).

PIC formation can also be regulated by overexpression of eIF5 or its mimic proteins 5MP1/2. When present in excess, these proteins can bind to eIF2 and sequester it from the 40S ribosome (144, 145). Similarly to eIF2α phosphorylation, binding of eIF5 or the 5MPs reduces global translation but enhances that of uORF containing transcripts including ATF4 (146). This mechanism appears to be important for the malignant properties of some cancer types such as fibrosarcoma and salivary mucoepidermoid carcinoma (147).

1.5.2.3.3 eIF3, connecting the eIF4F and pre-initiation complexes eIF3 is a multi-subunit complex that binds to eIF4G and the PIC, thus bridging mRNAs and the 40S ribosomal subunit and allowing scanning to occur (13). Theoretically, increased eIF3 levels should help promote this bridging and therefore the efficiency of translation initiation. However, several studies on eIF3 paints a more complex picture. Indeed, when overexpressed or knocked down in immortalized cells, individual subunits display oncogenic properties, while others behave more like tumor suppressors (24). Similarly, levels of eIF3a, b, c, h, i and m are increased in a variety of cancer types, while reduced expression of eIF3e and f have been reported. These associations should be interpreted with caution, as many rely on measurements of mRNA concentrations in patient samples, which may not reflect protein levels, or on RNAi-mediated depletion of eIF3 subunits to levels much lower than the minimum required to support translation, as discussed in depth elsewhere (24). Further complicating matters is the possibility of non-translational roles of certain subunits, such as eIF3a, which has been reported to bind to components of the cytoskeleton (148, 149), or eIF3f and i, which have been proposed to regulate signal transduction pathways (150, 151). Perhaps a better understanding of the different cancer-related roles of eIF3 subunits can be gleaned from recent studies highlighting their translation-specific roles. eIF3 can bind to mRNA structures in the 5’UTR of such cancer-relevant mRNAs as c-Jun and Btg1, resulting in translational activation of c-Jun and repression of Btg1; these opposing effects could be due to different eIF3 subunits (152). In fact, the same group has proposed that eIF3d specifically binds to the cap of the c-Jun mRNA and described an entirely novel model of specialized cap-dependent translation (153). In addition, loss of eIF3d and eIF3e has been linked to the translation of a metabolic program promoting the so-called Warburg effect. eIF3e has also been proposed to promote the phosphorylation of eIF4E (62). In this last case, it is difficult to reconcile the supposed tumor suppressive properties of eIF3e with the oncogenic aspects of eIF4E phosphorylation.

1.5.2.3.4 Alternative initiation In stress conditions leading to eIF2α phosphorylation, translation is impaired but not abrogated. Recent publications have shed light on an alternative mechanism of recruiting initiator tRNAs to

24 ribosomes and the initiation of translation, which is dependent on eIF2A (154). This protein promotes initiation at non-canonical CUG and UUG codons, which encode leucine, leading to questions as to which tRNA species is used to initiate translation. While some groups have reported initiation with leucyl-tRNA, others have indicated that the eIF2A-initiator-tRNA interaction is much stronger (155-158). Notwithstanding this issue, eIF2A appears to play a crucial role in the translation of oncogenic mRNAs including c-Src, BiP, N-Ras, CD44, Ki67, Nrp1 and Rac1 (155, 156, 159). However, this role is limited to stress conditions and appears particularly important to cancer cells, as depletion of eIF2A has no effect on the proliferation of normal cells, but severely impedes tumorigenesis and the proliferation of cells with phosphorylated eIF2α (155, 156). This characteristic argues for therapeutic targeting of eIF2A, though no such therapy has yet been developed.

1.5.2.5 Translation elongation and termination While much of the scientific focus has been on translation initiation, oncogenic changes in elongation and termination are emerging as important players in cancer. For example, a dominant role for the inhibition of elongation via the mTOR-S6K-eEF2K pathway has been demonstrated for intestinal tumor formation (92). Furthermore, the availability specific tRNA isoacceptors appears to play a role in tumorigenesis. Indeed, the speed of amino acid incorporation during the elongation phase is dependent on the availability of the corresponding charged tRNA (160). Several studies have reported distinct translational programs, where proliferating/cancer cells express an array of tRNAs optimized to correspond to the codon usage of pro-proliferative mRNAs, which is different than the codon usage and corresponding tRNAs of differentiated cells (161-163). Hence, in cancer cells the repertoire of available tRNAs is thought to be rewired such that the species required for the translation of oncogenic mRNAs are present at sufficient levels. In addition, elongation can also be deregulated in cancer via programmed -1 ribosomal frameshifting (-1 RPF), a process by which sequence elements force elongating ribosomes back by one base, leading to frameshifts, pre-mature stop codons and nonsense-mediated mRNA decay. Indeed, oncogenic pathways such as the JAK-STAT signaling pathway may be disproportionately regulated by -1 RPF, suggesting that members of this

25 pathway are normally translationally repressed at the elongation step, which could theoretically be relieved in cancer cells (164). This may explain the oncogenic role of silent mutations inducing frameshifting in tumor suppressors (164).

Aberrant termination in the cancer setting has garnered limited attention. However, termination at premature stop codons occurring due to somatic mutations in tumor-suppressor genes is also a cancer driver (165). This process promotes mRNA decay, known as nonsense- mediated mRNA decay (NMD). mRNA degradation by NMD prevents the expression of truncated proteins resulting from premature stop codons, as these may have dominant negative properties or gain-of-function activities that would be detrimental to cells (165). However, when occurring in tumor suppressors, reduced expression due to NMD is itself detrimental and can result in transformation (165). NMD can be prevented using aminoglycosides or small molecule drugs that promote readthrough of premature stop codons. Clinical introduction of a small molecule inhibitor for the treatment of Duchene Muscular Dystrophy, known as Translarna (Ataluren) by PTC Therapeutics is approved in a number of countries (166, 167).

1.5.2.5 Factors regulating multiple steps of translation 1.5.2.5.1 eIF6 and eIF5A Two initiation factors also play definite, but confusing, roles at multiple steps of translation. The first is eIF6, an anti-association factor preventing aberrant interactions between the 40S and 60S ribosomal subunits. eIF6 must be displaced for the final step of ribosome formation in the nucleolus, a role emphasized by the finding that eIF6 is a key player in ribosomopathies, cancer syndromes characterized by aberrant ribosome synthesis (see below and (164)). In addition, eIF6 can theoretically promote 80S ribosome disassembly by preventing the re-association of post-termination 60S ribosomes, thereby shifting the equilibrium towards termination and preventing further rounds of initiation with prolonged sequestration (26). Thus, eIF6 may play multiple roles in deregulating translation. In addition to its role in ribosomopathies, aberrant eIF6 expression has been observed in colorectal and head and neck cancers, where it

26 accumulates in the nucleolus (168, 169). In contrast, reduced eIF6 levels have been shown to prevent oncogene-induced transformation, and delay lymphomagenesis (170, 171).

The second is eIF5A, which was originally described as an initiation factor important for the formation of the first peptide bond, but has since been found to play a role in the elongation of poorly translated tripeptides containing prolines, glycines and/or basic residues (25, 172-174). A role in translation termination has also been proposed based on the accumulation of stalled ribosomes at stop codons in cells lacking eIF5A (174, 175). There is much interest in this initiation factor, as both of its isoforms are overexpressed in a variety of cancers including pancreatic, hepatic, colon, lung and ovarian, and have been linked to the metastatic capacity of cancer cells (reviewed in (25)). As the only known mammalian protein containing a hypusine modification, which is a modified lysine, eIF5A is an attractive target, as its activity can be abrogated by inhibiting the enzymes catalysing the hypusination (176).

1.5.2.5.2 The MYC oncoprotein Simultaneous deregulation of several aspects of translation can also occur via amplification of Myc. MYC is a promiscuous transcription factor that can target up to 15% of the genome, including the translation machinery (177). MYC promotes the synthesis of rRNAs and tRNAs by stimulating the activity of RNA polymerases I and III (178, 179), and can activate the transcription of Eif2, Eif4e, Eif4a and Eif4g (180, 181) (182). As the Myc mRNA was one of the earliest identified eIF4E-sensitive targets (183), there exists an interesting feed-forward mechanism between the MYC transcription factor and the translational apparatus. In addition, MYC-dependent protein synthesis increases eIF2α phosphorylation and activates autophagy as a survival pathway important for tumor development. Thus, MYC comprehensively deregulates translation to promote its own expression, and likely, that of survival factors required to escape MYC-induced apoptosis, to the great benefit of the transformed cells in which it is overexpressed. As a result, MYC-dependent tumors are addicted to activated translation, which provides an therapeutic opportunity to target protein synthesis in patients characterized by the classically untargetable MYC (184).

1.5.2.5.3 The cancer ribosome Encompassing all of these possible oncogenic changes in translation initiation, elongation and termination are modifications in ribosomes themselves. A major boon to research on the importance of translation in cancer has been the discovery in the first years of the 21st century that inherited mutations in genes encoding ribosomal components or regulators thereof cause ribosomopathies, a family of syndromes leading to distinct pathological features including increased cancer susceptibility. The identity of the various mutated genes with their respective outcomes have been thoroughly reviewed elsewhere (164). Of note, most of these mutations are rarely observed in sporadic tumor cases, which present a different array of ribosomal defects. The basis for these discrepancies is unclear, as is the mechanism underlying the oncogenic effects of these mutations.

One hypothesis relies on observations that the stoichiometry of ribosomal components and rRNA modifications, such as methylation and pseudouridylation, varies in cancer cells. This suggests that individual ribosomes may possess unique modifications altering their ability to translate certain mRNAs with respect to others. “Cancer ribosomes” would therefore promote the translation of oncogenic mRNAs and/or restrict the translation of tumor suppressors. In support of this concept is the finding that disruption of dyskerin, the enzyme catalysing pseudouridylation, or of snoRNAs guiding dyskerin to rRNA sites, is common in cancer and can impair the translation of mRNAs encoding critical tumor suppressors such as p53 and p27 (71). Most recently, state of the art mass spectrometry to measure the absolute abundance of core ribosomal proteins, including those found mutated in ribosomopathies, has provided direct evidence of ribosome heterogeneity. Moreover, genome-wide ribosomal profiling of such heterogeneous ribosomes reveals that they preferentially translate distinct subsets of genes, including those critical for cellular proliferation, metabolism, and embryonic development (185). These findings support the notion that changes in ribosome composition may lead to specific pathologic features such as cancer, as a consequence of perturbations in the translation of specific networks of mRNAs.

Alternatively, the link between ribosomal components, ribosomopathies and cancer has been attributed to non-translational roles, mainly p53 stabilization (3, 186). These appear to play important roles in responding to nucleolar stress and regulating cell cycle progression. Thus, a sub-ribosomal complex composed of the 5S rRNA and ribosomal proteins RPL5 and RPL11, binds to and sequesters HDM2, resulting in p53 stabilization and cell cycle arrest. This complex forms when deregulated ribosome biogenesis leads to the accumulation of imbalanced ribosomal components, allowing the excess to inhibit proliferation until the proper balance is restored (3). Somatic loss of p53 signaling abrogates this effect, and is thought to be the cause of the cancer predisposition associated with ribosomopathies. A similar example relates to the ability of subsets of ribosomal proteins to modulate MYC expression, which in turn regulates nearly all the components of the translation machinery (187).

1.5.2.6 A variety of initiation factors with a variety of effects. There are many ways in which cancer cells can regulate translation, all with different potential effects that are incompletely understood. The eIF4F complex alone provides multiple options. The simplest model for eIF4F complex activity in cancer would predict that modulating the levels or availability of any of its subunits would lead to changes in eIF4F complex formation, with similar results. In support of this, eIF4E, eIF4G and eIF4A are all critical for the translation of some initiation factors such as cyclin D1 (101, 188-190). However, functional parity does not always appear to be the case. Thus, eIF4E and eIF4G overexpression are found in different cancer types, and only eIF4E levels appear to affect metastatic progression (101, 191). Furthermore, inhibiting or depleting different subunits yields different results: whereas eIF4E inhibition tends to be cytostatic, eIF4A inhibition is potently cytotoxic (106). These discrepancies can be attributed to the varying roles of individual eIF4F subunits: the eIF4E- eIF4G interaction is required for all cap-dependent translation, with eIF4A only being required to unwind stable 5’UTR structures; in contrast, eIF4G and eIF4A, but not eIF4E, are required for cap-independent translation mediated by an internal ribosome entry site (IRES). Hence, specificity can be achieved by modulating specific eIF4F subunits. Examples of this phenomenon

29 include the upregulation of eIF4G seen in locally advanced breast cancer to promote IRES- mediated translation of VEGFA (191), or observed in response to genotoxic stress to promote the translation of factors involved in DDR (192). Another example is that of mitochondrial- induced apoptosis downstream of eIF4A inhibition, but not eIF4E (see section on energetic stress). In light of this, the array of possible ways for cancer cells to deregulate translation must provide a wide range of benefits, as well as possible therapeutic strategies, which remain to be fully understood.

1.5.3 Selective advantages of deregulated translation (cancer outputs) 1.5.3.1 Proliferation and apoptosis Subsequent to the finding that eIF4E transforms fibroblasts, much research demonstrated the crucial role of translation in the regulation of proliferation and survival. Thus, several anti- apoptotic factors, cyclins and cyclin-dependent kinases have been shown to be regulated by eIF4E and many other initiation factors, a fact that has been extensively reviewed (100, 101, 193-195). While this has historically monopolized much of the research in the field, other aspects of cancer are also regulated at the level of translation (Figure 1.3). This is particularly striking when considering that at least one regulatory event, the phosphorylation of eIF4E, can promote neoplastic transformation and metastatic progression without affecting proliferation or survival (55, 97).

1.5.3.2 Angiogenesis Angiogenesis can be promoted by a variety of translational mechanisms. Thus, FGF2 and VEGFA levels have long been associated with eIF4E overexpression in human tumors (196, 197). But VEGFA also possesses an IRES allowing for regulation by eIF4G (191) and uORFs that confer dependency on eIF2α phosphorylation (198), among the many layers of post-transcriptional regulation of VEGFA (199). This renders cancer cells particularly adaptable to oxygen deprivation, as seemingly all translational roads lead to neo-angiogenesis. Similarly, the major factor responsible for the response to hypoxia, HIF1α, can be translationally regulated by cap- dependent and independent mechanisms. Thus, in the context of hypoxia-induced mTOR

30 inhibition, 4E-BPs sequester eIF4E, promoting IRES-dependent expression of VEGFA and HIF1α (191, 200), in a process that may involve YB1 (201). However, in cells overexpressing eIF4E, or displaying hypoxia-resistant mTOR activation, sufficient eIF4E is available to promote cap- dependent translation of the mRNAs encoding these factors (202-204). Interestingly, HIF1α binds to the Eif4e promoter to promote its transcription, suggesting the possibility that the response to hypoxia could switch from an initial cap-independent mechanism, to a cap- dependent one (203). This model is supported by the fact that eIF4E promotes the translation of mRNAs encoding key hypoxia-response factors including the major hypoxia-related transcription factor, HIF1α, and growth factors promoting vascularization such as FGF2 and VEGFA (196, 197, 202). A more controversial report proposes that eIF4E2, also known as 4EHP, serves as an alternative cap-binding protein and recruits ribosomes via an alternative initiation complex during hypoxia (205). Of note, 4EHP is expressed at lower levels and possesses much poorer affinity for the cap than eIF4E (206, 207); therefore, how 4EHP could compete with eIF4E for cap-binding remains to be demonstrated. Specialized translation by non-canonical cap-binding proteins has also been proposed for eIF3d and the c-Jun mRNA (153).

1.5.3.3 Stress response These mechanisms allow for VEGFA translation in a variety of stress conditions, particularly hypoxia, resulting in angiogenesis to increase tumor vascularization and overcome the oxygen deprivation. Many other stresses are encountered by cancer cells on the road to malignancy, including oncogenic stress, nutrient deprivation, and oxidative stress, with translational deregulation of the response providing cancer cells with the means to thrive in these highly stressed conditions (132, 208). The most striking example is that of eIF2α phosphorylation, which allows for the increased translation of uORF-containing stress-response mRNAs, including prototypical ATF4, CHOP, and many others. In fact, up to 49% of the transcriptome, and essentially all mRNAs translated in stress conditions, have been reported to include uORFs, and disproportionately encode proteins involved in pathways allowing cancer cells to adapt to their environment (208-210). Though beneficial in response to acute stress, the associated inhibition of general translation, if persistent, eventually causes cell death (132, 208). Cancer cells must

31 therefore develop mechanisms to dampen this global effect, either by fine tuning the amount and duration of eIF2α phosphorylation, or by dissociating the pro-apoptotic effects from the stress response. For example, eIF2α phosphorylation promotes the translation of its own phosphatase CreP, resulting in a feedback inhibitory loop (210). Furthermore, IRESs, mRNA methylation and eIF2A can help maintain protein synthesis when cap-dependent translation is inhibited, allowing cancer cells to survive the pro-apoptotic consequences of eIF2α phosphorylation (132, 141-143). However, further research is needed to better understand how cancer cells adapt translational control during prolonged stress. Recent advances in how translation modulates the cancer-specific stress response are delineated below.

1.5.3.4 Oncogene Induced Senescence (OIS) A critical barrier to neoplastic transformation is the coupling of oncogenic stress with the pro- proliferative properties of oncogenes, resulting in senescence or apoptosis. This is related to accumulating DNA damage due to hyperactive proliferative signaling, and activation of the p53/p21 and p16INK4A tumor-suppressor pathways and growth arrest (211-213). Overexpression or hyperactivation of eIF4E was previously reported to act as a classical oncogene by inducing senescence in mouse embryonic fibroblasts and in B cells (99, 214). In contrast, recent studies using breast cancer models describe eIF4E as an exception to this rule, as its overexpression promotes sustained proliferation without inducing OIS (215, 216). While DNA damage occurs in cells overexpressing eIF4E (215), they benefit from the concomitantly increased translation of DNA repair factors such as BRCA1 (217), in addition to factors promoting survival and cell cycle progression (216, 218). As a result, they are able to overcome the anti-proliferative and pro-apoptotic signals associated with DNA damage and, thus, uncouple OIS from their oncogenic signaling (215). Beyond this apparent cell-type specificity of eIF4E-related OIS, it is well established that aberrant eIF4E expression can counteract RAS- mediated OIS and apoptosis induced by c-MYC, cooperating with these oncogenes to promote neoplastic transformation (99, 219, 220). Alternatively, cells undergoing transformation can evade OIS and oxidative stress via the eIF2α-ATF4 axis (131, 221, 222). Mechanistically, eIF2α phosphorylation promotes ATF4 mRNA translation, which in turn suppresses the transcription

32 of the major senescence inducing gene Cdkn2a (131, 222). Consequently, levels of p16INK4 and p19ARF decrease, preventing senescence. The implication of both eIF4E and eIF2 in the escape from OIS is demonstrated by a proteome-wide study of ubiquitinylation by Bengsch et al., where translational control was identified as critically important in this process (223). Thus, cancer cells, even in pre-neoplastic stages, deregulate translation to evade senescence and achieve transformation.

1.5.3.5 Metabolism Low levels of oxygen and/or nutrients can prevent proper energy production by cancer cells, as can mitochondrial damage and chemical inhibitors of oxidative phosphorylation (224). The link between translation and energy status is mediated by AMPK, which is activated by an increase in the AMP/ATP ratio, leading to phosphorylation of TSC2 and inhibition of translation via the mTOR/4E-BP axis. Phosphorylation of eIF2α downstream of AMPK has also been implicated, specifically in the context of drugs targeting NAD+ synthesis (225). Thus, energy deficits lead to the inhibition of translation and energy conservation. In turn, many mitochondrial proteins are regulated at the level of translation (226, 227). Interestingly, Gandin et al. demonstrated that translational co-regulation of these mitochondrial proteins and anti-apoptotic proteins constitutes a critical survival mechanism. As synthesis of proteins maintaining the integrity of the outer mitochondrial membrane (e.g. BCL2, MCL1, BIRC5) is decreased due to mTOR inhibition, mitochondrial activity is coordinately downregulated by preventing the synthesis of key electron transport chain components (e.g. ATP5O, ATP5G1, NDUFS6, UQCC2) (105, 226). Such co-regulation allows cancer cells to rapidly adapt to changing energetic states, but may also confer a targetable weakness, due to differences in the 5’UTRs of the two groups of mRNAs. Oxidative phosphorylation-related mRNAs possess short 5’UTRs enriched for translation initiator of short 5′ UTR (TISU) elements, whereas survival-related mRNAs possess long, structured 5’UTRs (105). Thus, only the latter group requires the helicase activity of eIF4A to unwind 5’UTR secondary structures. This key difference may explain why mTOR inhibitors are cytostatic, whereas eIF4A inhibitors are cytotoxic, as mitochondrial activity continues despite decreased ability to maintain membrane integrity (105). Another important mechanism

33 linking energy status to translation has been reported, whereby energetic stress leads to alternative transcription start site selection, altering the 5’UTRs of a wide array of transcripts and therefore their potential for translational control (228). Many regulated mRNAs are themselves involved in translation, thus adding to the complexity of the interplay between energy status and translation. For example, under glucose starvation, the gene encoding poly-A binding protein (PABP) is transcribed from an alternative promoter, resulting in a transcript with a shorter 5’UTR lacking autoinhibitory sequences and displaying increased translation (228). Thus, there are several mechanisms that we are only beginning to uncover by which cancer cells can adapt translation to their energetic requirements. Whether this can be targeted in cancer patients remains to be seen, but it is an area of active investigation (102).

1.5.3.6 Drug Resistance One of the most studied and clinically dire attributes of translational deregulation is the ability to confer drug resistance on cancer cells. There are numerous reports of resistance to chemotherapy, radiation and targeted therapies being mediated by alterations in the translational machinery (95, 229-231), which has been called a “nexus of resistance” (231). Some of the most recent observations include phosphorylated eIF4E-mediated resistance to radiation, DNA damage and alkylating agents (232-234), as well as resensitization of ovarian cancers to carboplatin by mTOR kinase inhibitors (217). Similar findings have been observed for eIF2α phosphorylation, in that use of salubrinal, a phosphatase inhibitor that prevents eIF2α dephosphorylation, enhances the efficacy of doxorubicin (235) and the proteasome inhibitor bortezomib (139). These effects are thought to be mediated by increased translation of mRNAs encoding DNA repair and survival factors in the case of deregulated eIF4F complex formation (95, 217, 229-231), and to the pro-apoptotic properties of long-term maintenance of eIF2α phosphorylation in the case of salubrinal (139, 235).

1.5.3.7 Emerging advantages of deregulated translation While the impact of translation on proliferation, survival and the stress response has been known for decades, its involvement in other aspects of tumor biology are only now being

34 uncovered. Thus, a variety of translation initiation factors, most notably eIF4E and eIF4G, have been shown to promote the metastatic potential of cancer cells by promoting the translation of pro-metastatic factors such as MMPs, integrins, transcription factors involved in epithelial-to- mesenchymal transition, small GTPases involved in migration, survival factors and DNA damage and repair (DDR) factors (97, 108, 191, 192, 236-242). In addition, eIF4E hyperactivation in cancer cells increases the translation of key anti-oxidant proteins such as ferritin and members of the glutathione pathway to sustain cancer cell survival by reducing reactivate oxygen species (ROS) levels (109). Interestingly, a cytosine-rich motif known as CERT is present in the 5’UTRs of these anti-oxidant mRNAs and is functionally important to sensitize these mRNAs to eIF4E expression levels (109). Translational regulation of the response to ROS may be particularly important to maintain translation in conditions of oxidative stress, as cysteine oxidation of components of the translation machinery is particularly sensitive to ROS (243). Protein synthesis thus provides a crucial means for cancer cells to disrupt a variety of processes important for all steps of tumor biology.

1.5.4 Inhibitors of translation Considering the importance of translation for many aspects of cancer cell biology, it is not surprising that many inhibitors of the translation machinery have been developed to treat cancer patients (Figure 1.5). The following section describes their mechanisms of action and clinical use.

1.5.4.1 mTOR inhibitors - Rapamycin and its analogs (Rapalogs) Rapamycin (sirolimus) is a naturally occurring macrolide produced by Streptomyces hygroscopicus, which was first described as an antifungal agent in 1975 (244). Yeast TOR kinases, and subsequently mTOR, were identified as the sole targets of rapamycin (245, 246). Rapamycin binds to the FKBP-rapamycin-binding (FRB) domain of mTOR in a complex with the immunophilin FKBP12 (FK506-binding protein) (246). This is thought to induce conformational changes that weaken the Raptor:mTOR interaction (247, 248), thereby inhibiting mTORC1 (249- 252). In addition, rapamycin:FKBP12 appears to induce steric changes in the FRB domain that

35 restrict substrate access to the catalytic site of mTOR (249, 252). The effects of rapamycin also seem to depend on the nature of the residues surrounding substrate phospho-acceptor sites, which partly reconciles why this drug inhibits phosphorylation of some, but not all mTORC1 phosphorylation sites (253, 254). Whereas mTORC2 is insensitive to acute rapamycin treatment (250, 251), prolonged rapamycin treatment suppresses mTORC2 levels in a subset of cell lines and in the liver, possibly as a consequence of newly synthesized mTOR molecules being sequestered by rapamycin:FKBP12 complexes (255, 256).

Several rapamycin analogs (rapalogs) were generated (everolimus, temsirolimus and ridaforolimus) to improve upon its pharmacodynamics properties, which exert anti-neoplastic activity in cancer cell lines and mouse models (257). All are in use in the clinic or in clinical trials (258). However, the efficacy of rapalogs in the treatment of human cancers has been less than expected (259). This has been attributed in part to incomplete inhibition of the 4E-BPs as well as to activation of AKT via disruption of mTORC1-S6K-PI3K and mTORC1-Grb10-PI3K negative feedback loops (260-263). In addition, rapalogs activate MAPKs in a PI3K-dependent manner (264). Thus, there are several mechanisms of resistance to this class of mTOR inhibitors.

1.5.4.2 Second-generation mTOR inhibitors More recent therapeutic strategies aimed at mTOR have addressed some of the shortcomings associated with rapamycin. The activation of AKT can be avoided by using dual specificity inhibitors that target both mTOR and PI3K, which is the main AKT-activating kinase (258, 265). The clinical applicability of these dual inhibitors in cancer is unclear, and the results of current ongoing clinical trials of PI-103, NVPBEZ235 and other PI3K/mTOR inhibitors are not available (258). Active-site mTOR inhibitors (asTORi, also called TORCi and TORKinibs) have also been synthesized (259). These potently inhibit both mTORC1 and mTORC2 (266-268). Since mTORC2 can activate AKT by phosphorylating it on S473 (269, 270), asTORi suppress AKT signaling (259) and exhibit stronger anti-neoplastic effects as compared to rapalogs (259). However, it has been shown that loss of mTORC2 activity does not reduce the anti-proliferative activity of the asTORi PP242 or Torin1 (266, 267), therefore demonstrating that the inhibition of mTORC1 is

36 responsible for the anti-proliferative effects of asTORi. Considering that rapamycin strongly and sustainably inhibits phosphorylation of some (e.g. S6Ks), but not all (e.g. 4E-BPs) mTORC1 substrates (271), the superiority of the anti-neoplastic effects of asTORi relative to rapamycin might be a consequence of asTORi drastically inhibiting “rapamycin-resistant” mTORC1 outputs, including 4E-BP phosphorylation (259, 266, 267).

In preclinical models, asTORi have shown promising results, including bioavailability, anti- metastatic properties, increased potency compared to rapamycin in vivo, while displaying specificity for cancer over normal cells (76, 272-274). However, asTORi cannot completely escape the resistance mechanisms inherent to targeting of upstream signaling molecules. Several studies indicate that eIF4E and/or 4E-BP expression levels affect the efficacy of PI3K and mTOR inhibitors (275-280). These studies demonstrate that mTOR-targeted therapies would likely be ineffective in tumors that exhibit an elevated eIF4E/4E-BP ratio (reviewed in (281)). Mechanistically, this is explained by the inability of mTOR inhibitors to effectively suppress eIF4F assembly; with translation of “eIF4E-sensitive” mRNAs proceeding unabated (275). These findings suggest that eIF4E/4E-BP ratio in a tumor could be used as a stratification marker for patients receiving mTOR-targeted therapies (275), since expression of these markers varies widely – a situation that has been documented in several cancers, including prostate and breast (116, 282). Additional mechanisms of resistance to mTOR inhibition include a switch from cap- dependent to cap-independent translation (283) and compensation of reduced mTOR signaling by activation of MAPKs (264).

Finally, the biguanide metformin has been shown to significantly reduce the risk of cancer in diabetic patients (284-287). Metformin suppresses mTOR (288) and represses translation of cancer-related mRNAs, though the subsets of mRNAs targeted by metformin and other mTOR inhibitors are only partially overlapping (227). Currently, many clinical trials are repurposing metformin for cancer therapy, though initial results are disappointing (289). 1.5.4.3 MNK inhibitors

Several small molecule inhibitors targeting MNKs have been described, including CGP57380 and cercosporamide (290-292). These reduce eIF4E phosphorylation and repress neoplastic growth in cell culture (290). While CGP57380 is ineffective in vivo, cercosporamide was shown to drastically decrease the growth of lung colonies in an experimental metastasis assay, suggesting a possible use for MNK inhibitors in the treatment of metastatic cancers (290). However, both compounds exhibit significant off-target effects (290, 293). To address this problem, novel strategies for targeting MNKs have been developed. Several 5-(2-(Phenylamino)pyrimidin-4- yl)thiazol-2(3H)-one derivatives have been shown to take advantage of features unique to the catalytic domain of the MNKs: the presence in the magnesium binding site of a DFD- rather than the typical DFG-motif seen in other kinases, three atypical insertions in the kinase domain and a propensity to crystallize in the inactive conformation. This latter characteristic, unusual for a kinase, results in the exposure of an additional hydrophobic pocket which can be targeted for increased specificity (294, 295). Other strategies are based on resorcylic acid lactone analogues that chemically react with cysteines present in the catalytic domain (296), and retinoic acid metabolism blocking agents (RAMBA) which indirectly lead to MNK1 degradation and decreased eIF4E phosphorylation in breast cancer cell lines (297).

While inhibiting MNKs could serve as a means to prevent resistance to other chemotherapeutic drugs including rapamycin, gemcitabine and herceptin (118, 298, 299), resistance to MNK inhibitors remains unexplored; indeed, determinants of sensitivity to anti-MNK therapy are unknown. However, the position of MNKs downstream of MAPKs suggests that at least some of the mechanisms of resistance to MEK inhibitors (reviewed in (300)) may be relevant to MNKs.

1.5.4.4 Inhibitors of eIF4E-cap interaction The ability of eIF4E to cause neoplastic transformation requires its cap-binding activity, since overexpression of an eIF4E mutant defective in cap-binding is not tumorigenic (113). Cap analogs have long been used in in vitro studies of eIF4E function, however they suffer from poor permeability and stability in vivo (301). To circumvent this problem, the design of pro- drugs with desirable pharmacokinetic properties has been undertaken. The N-7 benzyl

38 guanosine monophosphate tryptamine phosphoramidate pronucleotide 4Ei-1 was reported to inhibit cap-dependent translation and EMT in zebrafish (302), and cause chemosensitization of lung cancer cells to treatment with gemcitabine (303). High-throughput screening of chemical libraries for effective, bioavailable cap mimetics is ongoing (304, 305). More recently, delivery of traditional cap analogs has been achieved in vivo using virus-like particles, opening new possibilities for eIF4E-targeting in cancer (306).

Ribavirin was reported to be an eIF4E-cap inhibitor (307), but this has been disputed (308, 309). A clinical trial reported benefits in acute myeloid leukemia patients (310), but the mechanism of action is unknown and can be attributed to translation-independent biological activities inherent to ribavirin (311).

1.5.4.5 Inhibitors of eIF4E-eIF4G interaction Another promising strategy to target the translational machinery is to interfere with the assembly of the eIF4F complex. High-throughput screening of chemical libraries identified 4EGI- 1, 4E1RCat, and 4E2RCat as inhibitors of the eIF4E-eIF4G interaction (312, 313). These molecules effectively downregulate the translation of eIF4E-sensitive mRNAs and have shown promise in pre-clinical models (312, 314). Intriguingly, although 4EGI-1 abrogates binding of eIF4E to eIF4G, it does not prevent binding of 4E-BPs to eIF4E (312). Recently, structural studies of 4EGI-1 in complex with eIF4E have revealed that this compound binds to a hydrophobic pocket distal to the eIF4G-binding site, causing localized conformational changes that result in allosteric inhibition of eIF4E:eIF4G interaction (315). 4EGI-1 also inhibits eIF4E-independent translation, possibly due to activation of stress response pathways (316, 317). An alternative means of disrupting the eIF4E:eIF4G interaction is through the fusion of 4E-BPs to the ligand of a cancer-specific cell surface receptor. For example, 4E-BP1 fused to an analog of gonadotropin- releasing hormone prevented eIF4F complex formation and inhibited tumor growth in a mouse model of ovarian cancer (318). Taken together, these results demonstrate the potential clinical value of targeting the eIF4E:eIF4G interaction directly.

1.5.4.6 Inhibitors of eIF4A Hippuristanol, Pateamine A, and silvestrol (rocaglamides) are eIF4A inhibitors which suppress translation (319). Hippuristanol belongs to a family of polyoxygenated steroids (320), which bind to the carboxy-terminal domain of eIF4A. It allosterically prevents eIF4A from interacting with RNA and blocks its helicase activity, both in its free form and as part of eIF4F (320). Pateamine A and the rocaglamide silvestrol increase the ATPase, RNA-binding, and helicase activities of eIF4A (321, 322). Both compounds appear to act as chemical inducers of dimerization, and increase the RNA-binding affinity of eIF4A to RNA in a non-sequence dependent manner, resulting in its depletion from the eIF4F complex (321). Pateamine A is an irreversible inhibitor of protein synthesis, likely the consequence of covalent inhibition of eIF4A, and is therefore very toxic in vivo (321, 323), though better tolerated derivatives have been developed (324, 325).

All three eIF4A inhibitors show pre-clinical efficacy in various cell and mouse models (190, 322, 324-327), with silvestrol demonstrating the highest potency in vivo (190). That inhibition of eIF4A is the mechanism by which silvestrol exerts its anti-proliferative properties on cells has been demonstrated by the identification of silvestrol-resistant eIF4A mutants that can rescue this effect (328). As expected for an eIF4F inhibitor, the translation of mRNAs harboring increased secondary structure in their 5’ UTR are more sensitive to inhibition by silvestrol (190, 329-331).

The demonstration that the eIF4A inhibitors can re-sensitize lymphomas to DNA-damaging agents in tumors overexpressing eIF4E suggests that directly targeting the eIF4F complex can overcome resistance mechanisms described previously that lead to increased eIF4E availability or expression. Interestingly, one of the barriers to development of silvestrol per se as an anti- neoplastic agent is that resistance can be mediated by overexpression of ABCB1/P-glycoprotein; structure-activity relationship studies are underway to overcome this limitation (329, 332, 333).

1.5.4.7 Inhibitors of Ternary complex formation Depending on the stimulus, intensity and duration, eIF2α phosphorylation can promote cell survival or have a deleterious effect on cell fate (334). A mutant of eIF2α which cannot be phosphorylated transforms NIH 3T3 cells (335), whereas sustained phosphorylation induces apoptosis (336). Therefore, increasing eIF2α phosphorylation is an attractive strategy to treat cancer (337). One way to achieve this is to activate the kinases residing upstream of eIF2α. BTdCPU, and related N,N'-diarylureas, promote eIF2α phosphorylation via the intracellular kinase HRI and show promising effects in vitro and in vivo (138, 338). Another strategy is to inhibit the dephosphorylation of eIF2α using phosphatase inhibitors, such as salubrinal (339). There is in vitro data suggesting a synthetic lethal relationship between salubrinal and the proteasome inhibitor bortezomib (139) and this is the basis of a clinical trial combining salubrinal with the proteasome inhibitor carfilzomib (NCT01775553). However, an important consideration in strategies aimed at increasing eIF2α phosphorylation is its pro-survival function in response to stressors, such as hypoxia and nutrient deprivation (340, 341). Dual effects of stimulators of eIF2α phosphorylation are exemplified by guanabenz, a compound that binds to protein phosphatase 1 and inhibits stress-mediated eIF2α dephosphorylation. Guanabenz was shown to promote the survival of HeLa cells under conditions of toxic ER stress (342), but inhibited tumor growth in a breast cancer mouse model (140). One possible means of avoiding the potentially detrimental effects of compounds like guanabenz would be to directly target TC formation, the feasibility of which has been demonstrated using a brominated derivative of fluorescein, NSC119889. This inhibitor prevents binding of tRNAMet to eIF2 in vitro (343), however, in vivo efficacy of direct TC inhibitors has yet to be established.

1.5.4.8 Other inhibitors of translation A few other inhibitors of translation show promise in the treatment of cancer. A bacterial enzyme, asparaginase, catalyzes the hydrolysis of L-asparagine and at a lower rate L-glutamine (5% of asparagine rate) (344), and is used for the treatment of acute lymphoblastic leukemia and pediatric acute myeloid leukemia (345, 346). Asparaginase leads to depletion of L- asparagine and L-glutamine, which is accompanied by perturbations in amino-acid pools,

41 increased eIF2α phosphorylation via GCN2 and inactivation of mTORC1 (347). Another clinically- approved natural product that inhibits protein synthesis is homoharringtonine, which prevents the formation of the first peptide bond (348, 349) and is approved for the treatment of chronic myeloid leukemia (350). Transcription of ribosomal DNA by RNA polymerase I is often increased in cancer (351). The small molecule CX-5461 effectively inhibits RNA polymerase I (352). This causes accumulation of free ribosomal proteins, leading to disruption of nucleolar function and induction of p53-dependent apoptosis (353). CX-5461 has so far demonstrated antitumor activity in murine xenograft models (352). Another noteworthy inhibitor is GC7, which blocks the hypusination of eIF5A (354). This is particularly interesting because eIF5A is the only known protein to contain the amino acid hypusine (355). GC7-mediated inhibition of hypusination leads to apoptotic cell death and impaired tumor growth in a mouse model of melanoma (354).

1.5.5 Context specificity It is convenient to picture deregulated translation as a continuous increase in translation of tumor-promoting mRNAs, and those favouring characteristics that contribute to overall cancer progression, such as proliferation, survival, angiogenesis, migration, etc. This view is supported by the observations that some aspects of deregulated translation appear to be uniformly distributed across entire tumors and predictive of outcome (117, 356), as well as by a wealth of studies utilizing carefully controlled environments that focus on very specific processes, some of which have been cited throughout this review. While the former publications validate translation as an important therapeutic target, and the latter have been absolutely crucial in developing our understanding of the translational control of cancer, they do not accurately represent the incredible plasticity, nor the dynamic nature, of this process. Cancer cells are not perpetually proliferating, and migrating, and promoting angiogenesis; rather, they respond to programmed and microenvironmental cues that steer the translational response in specific, advantageous directions.

To complicate matters further, different cell types within a tumor are responsible for different aspects of tumor biology. This has been highlighted by the ever-widening understanding of

42 intra-tumor heterogeneity, as tumors are comprised of many sub-populations of cells of varying types and functions. These include immune cells, stromal fibroblasts, vascular endothelial cells, stem-like tumor cells known as tumor initiating or cancer stem cells that are rare but able to repopulate entire tumors and likely underlie metastasis, among others. Moreover, different regions of tumors and their micro-environment are exposed to varying concentrations of nutrients and growth factors and respond across a gradient of stresses and in different ways that are cell type dependent. Thus, translational control may vary enormously across tumor regions and different cell populations within individual tumors, and we are only beginning to understand what these differences are, how they affect cancer and how best to strategize their therapeutic targeting.

1.5.5.1 Cancer cell stemness The importance of translation in stem cells has long been appreciated, especially given the low transcriptional activity of these cells in embryonic and hematopoietic systems. More recently, translational regulation has been shown to be a major determinant in differentiation programs. Thus, the 4E-BPs are required to limit translation and ensure the maintenance of hematopoietic and embryonic stem cells (357, 358), whereas their loss promotes reprogramming to induced pluripotent stem (iPS) cells (359). Moreover, eIF2α phosphorylation promotes the maintenance of muscle stem cells (360), and ribosome biogenesis regulates neural stem cell self-renewal (361). Interestingly, DAP5 expression promotes developmental cell differentiation in normal tissues, purportedly by enhancing cap-independent translation that would circumvent inhibition by 4E-BPs and eIF2α phosphorylation (362, 363). While cancer cells are not stem cells per se, they have been shown to similarly rely on translational control. Thus, tumor initiating cells in a mouse skin cancer model display reduced protein synthesis, which is linked to aberrant uORF translation, and thus likely dependent of eIF2α phosphorylation (364). Phosphorylation of eIF4E has also been implicated in the stem-like phenotype of glioma stem cells, and in acute and chronic myeloid leukemia by promoting the translation of stemness factors such as β-catenin (365-367). These studies indicate that cancer stem-like cells are characterized by low protein synthesis compared with most cancer cells, and may be associated

43 with hypophosphorylation of 4E-BPs, or hyperphosphorylation of eIF4E or eIF2α. The spectrum of inhibitors of translation to which they respond may therefore be different than that of bulk tumor cells, which must be taken into consideration for potential cancer therapies.

1.5.5.2 Fibroblasts Among the non-transformed cell types present in tumors, fibroblasts are often the most prevalent. Their important roles at all stages of tumor development, growth and metastasis have been extensively studied and reviewed elsewhere (368). Of interest to the current work is the finding that cancer associated fibroblasts displaying a senescence-associated secretory phenotype (SASP) are among the most highly translationally active cells. These cells secrete a wide variety of cytokines and chemokines, growth factors and matrix proteins which require an enormous amount of metabolic activity for their production (368). Interestingly, defects in ribosome biogenesis reminiscent of ribosomopathies have been reported to induce SASP in fibroblasts (369). Specifically, overexpression of TIF-IA, resulting in increased rRNA transcription, and depletion of rRNA processing enzymes, results in sequestration of MDM2 in the nucleolus by ribosomal components and the activation of p53 (369). Whether this plays any role in the pathogenesis of ribosomopathies, or the switch from a hypo- to hyper-proliferative disease, remains to be investigated. Furthermore, as the senescence appears to be induced through p53 activation, it is unclear how protein synthesis is regulated in this context. In other models, mTOR has been shown to promote the translation of the SASP-related immune modulator IL1A, and the MK-2 kinase (370, 371). It is unclear whether mTOR also affects the general biosynthetic capacity of senescent fibroblasts, though translational activation of TOP mRNAs is possible.

1.5.5.3 Immune cells The importance of translation for immune cell populations is evident from the immune- suppressive effects of inhibitors such as rapamycin (372, 373). The role of mTOR in T cells, among others, is the subject of much ongoing investigation, in particular with respect to its importance in translation, metabolic reprogramming, differentiation and activation (374-376).

Furthermore, in a several disease states such as allografts, cancer and injury, mTOR appears to be important for the biology of myeloid derived suppressor cells (MDSCs); however, it is not always clear whether this reflects mTOR in the cells recruiting MDSCs, in the MDSCs themselves, or both (377-379). Despite this, little is known regarding translation regulatory events specific to immune cell populations overall, and specifically in the cancer context, although hints can be gleaned from research in other models. For example, decreased eIF4E levels have been reported to be important in the differentiation of T regulatory cells (380). eIF4E phosphorylation in a variety of immune cell populations also appears to be important for the synthesis of cancer relevant cytokines and chemokines such as TNFα and IFNγ (381-384), and for the development of experimental autoimmune encephalomyelitis (385). In neutrophils, our unpublished results demonstrate that eIF4E phosphorylation promotes survival via increased translation of anti-apoptotic proteins, thus promoting metastatic dissemination in a mouse model of breast cancer. Similarly, eFFECTOR Therapeutics has reported that a novel MNK inhibitor, eFT508, possesses immune-modulatory properties and are pursuing its development in Phase II clinical trials. This aspect is particularly intriguing, as it suggests that targeting eIF4E phosphorylation in both the cancer and immune compartments should improve patient outcome. Whether other means of inhibiting translation may be similarly useful or more potent is an intriguing direction that remains to be investigated.

1.5.6 Concluding remarks As described herein, the translation of mRNAs into proteins is a critical aspect of the regulation of gene expression and plays a major in cancer. Every month brings new discoveries into the extent of its involvement, with previously unknown factors regulating novel aspects of tumorigenesis, in unsuspected cell types. Much of the information summarized here was unknown at the start of my studies in 2010. While it demonstrates the dynamic nature of research on translation and sharpens my interest in the field, it may lead to confusion for the reader as to the pertinence of certain experiments performed. Thus, the contextual setting of the research described in Chapters 2 and 3 is delineated at the start of each chapter.

CHAPTER 2

PHOSPHORYLATION OF EIF4E PROMOTES EMT AND METASTASIS VIA TRANSLATIONAL CONTROL OF SNAIL AND MMP-3

2.1 CONTEXTUAL SETTING The field of translational control of cancer has enjoyed an extraordinary expansion in the last decade, as our understanding of its importance has broadened from promoting proliferation and survival to encompass every aspect of the hallmarks of cancer. The importance of the current work can therefore be best understood as part of this overarching theme of applying the known regulatory mechanisms of translation to new aspects of tumor biology. Considering this, a timeline of the relevant discoveries leading up to the experiments described in Chapter 2, and the following studies validating these findings, is presented here.

By the early 2000’s, the importance of translation in cancer was well established. Much of the focus was on eIF4E, which was considered an oncogene promoting survival, proliferation and angiogenesis, and leading to transformation in vitro and in vivo (194, 236). In 2004, the phosphorylation of eIF4E was demonstrated to be required for its ability to transform NIH 3T3 cells in vitro (114), which was rapidly confirmed in an in vivo model of lymphoma (113). To validate these findings, the Sonenberg lab developed a mouse model in which the phosphorylation site of eIF4E is mutated to alanine at the endogenous locus (55). The resulting mice (termed eIF4ES209A hereafter) were developmentally normal, born at Mendelian ratios and presented no overt phenotypes; however, they displayed a clear resistance to prostate cancer induced by loss of the PTEN tumor suppressor (55). Furthermore, mouse embryonic fibroblasts (MEFs) isolated from these mice were resistant to neoplastic transformation (55). Around the same time - in one case, in the same issue of PNAS - other studies validated these findings by demonstrating that inhibiting MNKs impaired xenograft and allograft growth, and that mice lacking MNKs were resistant to cancer development in the Lck-Pten lymphoma model (56, 290). Surprisingly, in many cases, abrogating eIF4E phosphorylation had little impact on proliferation or apoptosis, despite severely impeding focus formation, anchorage independent growth and tumor formation in mice (55, 56, 290). This suggested that other factors translationally regulated by eIF4E phosphorylation played important roles in tumorigenesis.

Clues as to what aspects of tumor biology, independent of proliferation and apoptosis, responded to translational control by eIF4E phosphorylation were provided by us and other groups. Thus, elevated phospho-eIF4E in patient samples was associated with more aggressive disease stages in prostate cancer, penile squamous cell carcinoma, nasopharyngeal carcinoma and non-small cell lung cancer (55, 116, 386-388). Metastatic lesions are usually the cause of death in these cancers, suggesting that the MNK/eIF4E axis may regulate the metastatic process. Furthermore, the role of eIF4E and the 4E-BPs in metastasis and patient survival was being established at this time (116, 237, 282, 389); it was thus plausible that phosphorylation be important for this activity of eIF4E. In line with this idea, we identified certain pro-metastatic factors as being controlled by phospho-eIF4E, notably the matrix metalloproteinases MMP-3 and MMP-9 (55). MMPs were known to cleave several component proteins of the extracellular matrix (ECM) to promote migration and invasion (390) and to induce epithelial-to- mesenchymal transition (EMT) (391). Furthermore, Grzmil et al. reported that MNK1 regulated the expression of SMAD2, thus affecting EMT in glioblastoma cells; however, this effect required 72h of MNK inhibition to be detectable, suggestive of secondary effects, and the involvement of eIF4E phosphorylation was unclear (392). An MNK inhibitor, cercosporamide, was also shown to inhibit the growth of experimental metastases derived from B16 melanoma cells; however, as the cells were first seeded in the lungs, followed by treatment, the impact of eIF4E on the metastatic process could not be assessed (290). These observations pointed to a role for eIF4E phosphorylation in metastasis, but direct experimental evidence for this link was lacking.

In the work presented in this chapter, we investigated the role of eIF4E phosphorylation in metastasis. We demonstrated that phosphorylation of eIF4E promotes invasion in transformed MEFs, as well as TGFβ-induced EMT in normal epithelial cells. We identified mRNAs regulated by eIF4E phosphorylation, which could mediate its pro-metastatic effects, including Snail and Mmp- 3. Importantly, we validated our findings in vivo using a metastatic mouse mammary tumor model. Taken together, these results demonstrated that eIF4E phosphorylation is a key event in the metastatic process.

2.2 RESULTS 2.2.1 eIF4E phosphorylation promotes migration and invasion To explore whether eIF4E phosphorylation plays a role in metastasis, we utilized a cell model that we had previously developed (55). MEFs were derived from mice bearing the eIF4ES209A mutant and their WT counterparts, and transformed with c-MYC and H-RASV12. These WT and eIF4ES209A transformed MEFs display similar proliferation, cell cycle progression and levels of apoptosis, yet eIF4ES209A MEFs possess reduced tumorigenic potential (55). We chose to use this model as it is not dependent on overexpression, and avoids targeting the MNKs, which phosphorylate additional proteins such as Sprouty2 (393), cPLA2 (394) and hnRNPA1 (395, 396). To investigate the role of phospho-eIF4E in metastasis, we studied WT and eIF4ES209A transformed MEFs in assays that measure metastatic potential: anchorage independent growth (397), random migration (398), transwell invasion (399) and colony outgrowth in matrigel (400). As previously reported (55), eIF4ES209A MEFs formed 2-fold fewer colonies when plated in agarose (Figure 2.1A). This effect was specific to eIF4E phosphorylation as treatment with the MNK inhibitor CGP57380 reduced colony formation in the WT MEFs in a dose-dependent manner, whereas eIF4ES209A MEFs remained insensitive to this treatment (Figure 2.1A). Phosphorylation of eIF4E also promotes migration, as eIF4ES209A MEFs displayed ~20% reduction in random migration speeds, as seen by time-lapse microscopy (Figure 2.1B). Accordingly, these cells traveled shorter distances than their WT counterparts (Figure 2.1C). Strikingly, invasion was severely impaired in eIF4ES209A MEFs, as their invasion index was reduced 5-fold in a transwell invasion assay (Figure 2.1D). Corroborating these findings, in a matrigel colony outgrowth assay, colonies of WT MEFs exhibited a branched morphology, indicative of their invasion into the basement membrane matrix, while eIF4ES209A colonies remained spherical (Figure 2.1E). Thus, eIF4E phosphorylation promotes in vitro characteristics that correlate with metastatic potential, prompting further investigation into its role in metastasis.

Figure 2.1. eIF4E phosphorylation enhances in vitro metastatic properties. (A), Anchorage independent growth: WT and eIF4ES209A MYC/RAS-transformed MEFs were plated in agarose and incubated for 2 weeks with the indicated concentration of CGP57380, and colonies of 8 or more cells were counted. (B-C), Random migration as monitored by time-lapse microscopy: migration speed (B) and representative migration paths (C) were determined using the Metamorph software. (D) Transwell invasion: invasion index is given as the percentage of cells having crossed the porous membrane of a Boyden chamber in the presence of a layer of matrigel versus in its absence. (E) Colony outgrowth: representative images of colony morphology for cells seeded in matrigel and incubated for 8 days. Error bars represent standard deviations (A, D) or standard errors (B). All results are representative of at least 3 independent experiments. Statistical significance determined by one-way ANOVA followed by Bonferroni's Multiple Comparison Test (A, D) or Student T-test (B); * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; N.S. = non-significant.

2.2.2 Phosphorylated eIF4E promotes the translation of metastasis-related mRNAs. To study the mechanism by which eIF4E phosphorylation favors pro-metastatic characteristics, the expression levels of the Y-box-binding protein 1 (YB1) were assessed in WT and eIF4ES209A MEFs. This protein had previously been identified as playing an important role in eIF4E- mediated invasion (76). However, YB1 levels did not vary between the two cell lines (Figure S2.1), suggesting that this protein does not mediate the effect of phospho-eIF4E on invasion. To identify candidate factors that promote the pro-metastatic phenotype of WT MEFs, mRNAs translationally regulated by eIF4E phosphorylation were analyzed. To achieve this, pathway analysis was performed on a previously generated dataset of mRNAs translationally regulated by eIF4E phosphorylation (55). Pathways significantly increased in WT cells were identified and clustered using the DAVID functional annotation database (401, 402). Enriched clusters included potentially metastasis-related functions such as chemotaxis, proteases and immune signaling, as well as a general cancer cluster and one corresponding to glutathione metabolism (Figure 2.2A). Clusters corresponding to plasma membrane proteins, carbohydrate-binding proteins, leucine-rich repeat-containing proteins and zinc-binding proteins were also identified. The full list of clusters generated by DAVID and the mRNAs grouped in each cluster are presented in supplemental material (Figure S2.2, S2.3). To validate these findings and further investigate pro-metastatic mRNAs regulated by eIF4E phosphorylation, polysome profile analysis was performed on transformed WT and eIF4ES209A MEFs serum-stimulated for 2h. qRT- PCR was used to monitor the distribution across a sucrose density gradient of mRNAs chosen for their relevance to invasion and metastasis, or because they were identified in the clustering analysis. There was no significant difference in global translation between WT and eIF4ES209A MEFs, as the polysome profiles were nearly identical (Figure 2.2B). Accordingly, the distributions of housekeeping mRNAs β-actin and Gapdh across the sucrose density gradient were similar (Figure 2.2C, D). In contrast, the mRNA encoding MMP-3, which had been previously identified as being sensitive to eIF4E phosphorylation (55), was significantly shifted toward light polysomes in eIF4ES209A MEFs (Figure 2.2E). This is indicative of regulation of translation initiation by eIF4E phosphorylation. Similarly, the polysomal distribution of Snail mRNA, an important transcription factor in the induction of EMT (403), was also significantly

52 shifted toward light polysomes in eIF4ES209A MEFs (Figure 2.2F). However, no differences were detected for other factors tested including the EMT marker vimentin, the EMT-related transcription factor TWIST, the metalloproteinase MMP-14 and the vascular endothelial growth factor member VEGFC (Figure S2.4). Our results indicate that eIF4E phosphorylation promotes the translation of mRNAs encoding regulators of EMT and invasion such as SNAIL and MMP-3.

In keeping with the translational repression of Snail and Mmp-3 in eIF4ES209A MEFs, the expression of SNAIL and MMP-3 protein was also repressed in these cells (Figure S2.1 and Figure 2.3A). Next, it was pertinent to demonstrate that SNAIL and MMP-3 are responsible for phospho-eIF4E’s pro-metastatic properties. To this end, we overexpressed eIF4EWT or eIF4ES209A in eIF4ES209A MEFs; only eIF4EWT restored SNAIL and MMP-3 levels and rescued the invasion defect of eIF4ES209A MEFs (Figure 2.3A-B). Similarly, overexpressing either Snail or Mmp-3 mRNAs lacking their regulatory 5’ and 3’UTRs led to an increase in the expression levels of their respective proteins, in fact, to levels higher than those seen in WT MEFs (Figure 2.3A), and increased the invasion of eIF4ES209A MEFs (Figure 2.3B). These experiments demonstrate that SNAIL and MMP-3 mediate the pro-invasive properties of phosphorylated eIF4E.

Figure 2.2. eIF4E phosphorylation promotes the translation of mRNAs involved in EMT/invasion and metastasis. (A), DAVID clustering of significantly enriched pathways from a previously generated list of phospho-eIF4E translationally-regulated mRNAs. Metastasis-related terms are in red boxes. (B), Polysome profile and qRT-PCR analysis of (C), β-actin, (D), GAPDH, (E), Mmp-3 and (F), Snail mRNA in polysomal fractions of Myc/Ras-transformed WT and eIF4ES209A MEFs. Levels of mRNA in each fraction are given as a percentage of the total. Error bars represent standard deviations. Results are representative of at least 3 experiments.

Figure 2.3. Overexpression of eIF4E or phospho-eIF4E targets rescues invasion. (A) Western blot analysis and (B) transwell invasion of Myc/Ras-transformed WT and eIF4ES209A MEFs overexpressing the indicated proteins relative to WT. Error bars represent standard deviations. Results are representative of at least 3 experiments. Statistical significance determined by one- way ANOVA followed by Bonferroni's Multiple Comparison Test; p values are given relative to eIF4ES209A cells + vector; * indicates p<0.05; N.S. = non-significant.

2.2.3 eIF4E is phosphorylated during TGFβ-induced EMT SNAIL and MMP-3 are important factors promoting cell invasiveness, metastasis and EMT (391, 403), therefore we investigated whether eIF4E phosphorylation could play a role in EMT. Since transformed WT and eIF4ES209A MEFs are inadequate for studying EMT, we employed a well- studied cell model of EMT, in which normal murine mammary epithelial cells (NMuMG) are treated with TGFβ. This results in an easily observable transition from an epithelial morphology to a mesenchymal morphology, and is accompanied by quantifiable changes in the expression of EMT markers (404). Treatment with TGFβ engendered a strong increase in eIF4E phosphorylation in a time dependent manner (Figure 2.4A). Increased phospho-eIF4E levels were accompanied by an increase in the mesenchymal markers N-cadherin, fibronectin and vimentin, as well as a decrease in the epithelial marker, E-cadherin (for a review on EMT markers, see (405)). Ostensibly, TGFβ controls the phosphorylation of eIF4E via non-canonical signaling leading to p38 and MAPK activation (406). Indeed, the phosphorylation and activation of MNK1 was preceded by that of its upstream kinases, p38 and ERK (Figure 2.4B). In addition, chemical inhibition of ERK or p38 activation, using U0126 or SB203580, respectively, reduced TGFβ-stimulated phosphorylation of eIF4E (Figure 2.4C), which was abrogated by a combination of both inhibitors (Figure 2.4C). NMuMG cells engineered to express a dominant-negative mutant of p38 (p38AGF) or MKK3 (MKK3AL) are defective in undergoing a TGFβ-induced EMT (407). TGFβ failed to stimulate phosphorylation of eIF4E when p38 activation was blocked in p38AGF- and MKK3AL-NMuMG cell lines, consistent with non-canonical TGF-beta signaling impinging on eIF4E (Figure 2.4D). TGFβ-induced eIF4E phosphorylation was dependent on the TGFβ receptor kinase activity because SB431542, an inhibitor of the TGFβ receptor kinase (408), blocked eIF4E phosphorylation (Figure 2.4E). Taken together, these results demonstrate that activation of TGF signaling, which is frequently dysregulated in breast cancer (409), induces eIF4E phosphorylation, and that elevated phospho-eIF4E correlates with TGF-induced EMT.

Figure 2.4. eIF4E is phosphorylated downstream of MAPK during TGFβ-induced EMT. Western blot analysis of (A) EMT markers and (B) MAPK signaling in a time-course experiment of NMuMG cells treated with 5ng/mL TGFβ. Western blot analysis of the effect of ERK and p38 MAPK inhibitors (C), expression of dominant-negative MAPK signal transducers (D), and TGFβ receptor inhibition (E) on TGFβ-induced eIF4E phosphorylation in NMuMG cells. Results are representative of at least independent experiments.

2.2.4 eIF4E phosphorylation promotes TGFβ-induced EMT To determine whether eIF4E phosphorylation is a driver or a side-effect of EMT induction, we used a chemical inhibitor of the MNKs, CGP57380 (292). Treatment with this inhibitor blocked TGFβ-stimulated eIF4E phosphorylation and SNAIL expression (Figure 2.5A), without affecting Snail mRNA levels (Figure S2.5). Importantly, CGP57380 abrogated the molecular and morphological changes associated with EMT. Specifically, this inhibitor blocked the upregulation of the mesenchymal markers vimentin, fibronectin and N-cadherin by TGFβ (Figure 2.5A, upper panel), and favoured the maintenance of tight junctions, as seen by ZO-1 expression, characteristic of an epithelial phenotype (Figure 2.5A, lower panel and Figure S2.6). The effect of pharmacologically targeting MNK activity on TGFβ-stimulated EMT was not due to inhibition of the Smad2 transcriptional pathway, since TGFβ treatment resulted in comparable phosphorylation of Smad2 in the presence or absence CGP57380 (Figure 2.5A). To provide further evidence for the role of eIF4E phosphorylation in EMT, we knocked down MNK1 by siRNA. MNK1 was targeted because it is the isoform that responds to MAPK signaling, while MNK2 is relatively insensitive to upstream signaling and maintains a basal level of activation (410). Knock down of MNK1 prevented TGFβ-stimulated eIF4E phosphorylation as well as the TGFβ-induced expression of SNAIL and vimentin (Figure 2.5B, upper panel) and maintained the expression of ZO-1 (Figure 2.5B, lower panel and Figure S2.6), further demonstrating that MNK1-mediated eIF4E phosphorylation promotes EMT. As seen with the MNK inhibitor treatment, the effects of MNK1 knock down in NMuMG cells were not due to changes in SMAD2 activation (Figure 2.5B), and Snail mRNA levels were unaffected (Figure S2.5). Consistent with experiments performed using MEFs, YB1 levels were unaffected by eIF4E phosphorylation in NMuMG cells (Figure 2.5A, B).

Considering that the MNKs have targets other than eIF4E, notably Sprouty2 (393), cPLA2 (394) and hnRNPA1 (395), we performed a knockdown/add-back experiment to investigate the impact of phospho-eIF4E on EMT in a MNK-independent model. We stably expressed either eIF4EWT or eIF4ES209A in MCF10A cells, followed by shRNA silencing of the endogenous eIF4E. The shRNA was specific to the human eIF4E and therefore did not inhibit expression of the

61 exogenous murine eIF4E (Figure 2.5C). Knockdown of eIF4E impaired SNAIL expression and blocked TGFβ-mediated EMT, as indicated by reduced induction of vimentin and fibronectin (Figure 2.5C). Strikingly, when endogenous eIF4E was knocked down, only eIF4EWT but not eIF4ES209A rescued SNAIL, vimentin and fibronectin expression (Figure 2.5C). Importantly, only cells expressing eIF4EWT, either the endogenous or exogenous form, expressed appreciable levels of SNAIL, vimentin and fibronectin (Figure 2.5C). In contrast, cells expressing the S209A mutant form of eIF4E had low amounts of the latter EMT markers, similarly to cells expressing low levels of eIF4E (Figure 2.5C). The effects we observe in the MCF10A model system indicate that defects in the MNK/eIF4E axis do not impinge upon canonical TGFβ signaling, since TGFβ- induced phosphorylation of Smad2 remained unchanged under all experimental conditions (Figure 2.5C). Thus, these experiments demonstrate a critical role for translational control by phosphorylated eIF4E in TGFβ-induced EMT.

2.2.5 eIF4E phosphorylation correlates with EMT and invasion in vivo We validated our findings in vivo in a widely used model to study EMT and metastasis. This model consists of a series of isogenic mammary cancer cell lines (67NR, 168FARN, and 4T07) that are progressively more aggressive and present an increasingly mesenchymal phenotype (411). Specifically, when inoculated into the mouse mammary fat pad, the 67NR cell line forms only non-invasive primary tumors, 168FARN cells are locally invasive and detectable in the lymph node, while the 4T07 cells metastasize to the lung. In mammary tumors formed by these cell lines, increased MNK1 activation and eIF4E phosphorylation coincided with the acquisition of invasive properties and with the expression of SNAIL, as this protein was low in 67NR tumors, but increased in locally invasive 168FARN and metastatic 4T07 tumors (Figure S2.7). The correlation between SNAIL expression, eIF4E phosphorylation and the acquisition of invasive properties of these cells, in vivo, provides further evidence for the importance of eIF4E phosphorylation in the metastatic process.

Figure 2.5. eIF4E phosphorylation is required for TGFβ-induced EMT. (A) Western blot analysis (upper panel) and immunofluorescence (lower panel) of EMT markers in NMuMG cells treated with 5ng/mL TGFβ, with or without 20μM CGP57380. (B) Western blot analysis (upper panel) and immunofluorescence (lower panel) of EMT markers in NMuMG cells treated with 5ng/mL TGFβ in the presence of scrambled siRNA or siMNK1. (C) Western blot analysis of EMT markers in MCF10A cells expressing the indicated shRNAs and eIF4E variants.

2.2.6 eIF4E phosphorylation promotes tumor onset and metastasis in PyMT mammary tumor model To assess the role of eIF4E phosphorylation in metastatic progression using a genetic model, eIF4ES209A mice were crossed with MMTV-PyMT mice. In this mammary tumor model, tumors metastasize to the lungs with 100% penetrance (412). eIF4ES209A-PyMT mice exhibited a significant delay in tumor onset, developing palpable tumors on average 2 weeks later than their WT counterparts (Figure 2.6A), confirming the importance of eIF4E phosphorylation in tumorigenesis (55). The penetrance of mammary tumor development was also reduced in eIF4ES209A-PyMT mice, as approximately 10% percent remained tumor-free. Nonetheless, once established, both WT and eIF4ES209A tumors grew at similar rates (Figure 2.6B). End-point tumors displayed similar proliferation, as evaluated by ki67 staining of tumor sections (Figure 2.6C), and similar apoptosis, as measured by caspase 3 activation (Figure 2.6D). Importantly, the metastatic potential of eIF4ES209A tumors was reduced, as eIF4ES209A-PyMT mice displayed a two-fold reduction in lung metastases as compared to WT-PyMT mice (Figure 2.6E, F). Metastatic burden was assessed at equivalent tumor burden rather than at a defined age; it therefore does not reflect the late onset of eIF4ES209A tumors. Thus, eIF4E phosphorylation promotes the metastatic properties of eIF4E, in addition to its tumorigenic activity. Based on our findings that eIF4E phosphorylation promotes the translation of mRNAs encoding SNAIL and MMP-3, we analyzed the expression of these factors by IHC in tumor samples from PyMT-WT and PyMT-eIF4ES209A mice. Phosphorylation of eIF4E was elevated in WT tumors, as expected due to activation of the MAPK pathway by the PyMT oncogene (413), but remained undetectable in eIF4ES209A tumors (Figure 2.7A). SNAIL and MMP-3 exhibited localized expression patterns in both WT and eIF4ES209A tumors (Figure 2.7A). Indeed, as previously reported, these proteins were mainly expressed at tumor/stroma boundaries (414, 415). Importantly, WT tumors displayed increased SNAIL and MMP-3 expression (Figure 2.7A), resulting in a higher percentage of SNAIL and MMP-3 positive cells (Figure 2.7B,C). Taken together, these data support an important role for the phosphorylation of eIF4E in promoting invasion and metastasis through translational upregulation of its targets such as SNAIL and MMP-3.

Figure 2.6. eIF4ES209A mice are resistant to mammary tumor development and metastasis. (A) Onset and (B) growth of mammary tumors in WT and eIF4ES209A mice expressing the PyMT transgene. Quantification of (C) Ki67 and (D) cleaved caspase 3 immunohistochemical staining in tumor sections from WT and eIF4ES209A mice, displayed as percentage of positive cells using ImageScope’s nuclear stain algorithm. (E) Metastasis count and (F) representative lung images of WT and eIF4ES209A mice at experimental end point. Arrows point to lung metastases. Statistical significance determined by Student T-test; p-values are indicated, N.S.=non- significant. The number of mice used is indicated in the figure.

Figure 2.7. eIF4E phosphorylation correlates with expression of SNAIL and MMP-3 in MMTV- PyMT tumors. (A) Representative images of IHC analysis of eIF4E phosphorylation, SNAIL and MMP-3 expression in primary MMTV-PyMT tumors. (B) SNAIL and (C) MMP-3 quantification of IHC analysis of primary MMTV-PyMT tumors: indicated are percentages of positive pixels using ImageScope’s positive pixel count algorithm. Statistical significance determined by Student T- test; p-values are indicated. The number of mice used is indicated in the figure.

2.3 DISCUSSION In Chapter 2, we showed that eIF4E phosphorylation promotes metastatic progression, in addition to its role in tumor development. We provided mechanistic insight into the role of eIF4E phosphorylation in metastasis, by showing that phospho-eIF4E was required for the translation of Snail and Mmp-3 mRNAs, two factors that regulate EMT and invasion. Importantly, our investigation into the role of the MNK/eIF4E axis as an essential step during metastasis and TGFβ-induced EMT uncovered an unanticipated intersection of canonical and non-canonical TGFβ signaling to cooperatively promote EMT. We propose that a subset of mRNAs, transcriptionally induced by canonical TGFβ signaling, is better translated when eIF4E becomes phosphorylated via MNK1, which lies immediately downstream of the non-canonical TGFβ signaling pathway. Such co-regulation of transcription and translation has been evoked in other studies as well (416-418). For example, both ERK and β-catenin lead to transcriptional increases in c-Myc mRNA, and promote its translation by simultaneously activating cap- dependent translation; c-Myc in turn promotes the transcription of components of the translation machinery (419-421). A similar mechanism has been proposed for targets of NF-κB and Notch signaling (421).

Since our findings were published, they have been validated by other reports demonstrating similar EMT and invasion-promoting effects of MNKs and eIF4E phosphorylation in a variety of cell models (297, 422, 423). In addition, eIF4E phosphorylation has been correlated with poor outcome in melanoma and astrocytomas (356, 424). MNK1 had previously been linked to TGFβ signaling and was reported to promote the translation of SMAD2 (392). However, no differences in the level of SMAD2 expression or activation were observed in our experiments. Differences in cell type aside, the decreased SMAD2 levels upon MNK1 silencing observed by Grzmil et al. occurred after 72h and may be a result of secondary effects. All of the experiments here were performed after MNK1 siRNA silencing for 24h or less. These differences may explain the discrepancies between the two studies.

Our data demonstrated that eIF4E phosphorylation is important for EMT and invasion, which are early steps in the metastatic progression. This raised the interesting possibility of using eIF4E phosphorylation as a biomarker for tumors at high risk of metastasizing. However, it would be important to consider the levels of eIF4E phosphorylation in the context of total eIF4E expression, as well as expression and phosphorylation status of the 4E-BPs, as each of these markers can affect patient outcome, as seen in prostate cancer (116). Furthermore, based on results, we argued for the development of MNK inhibitors to prevent metastatic progression. At the time, the only bioavailable compound was cercosporamide, which had been shown to inhibit eIF4E phosphorylation and the growth of metastatic lung colonies in a B16 experimental metastasis assay (290). In this study, treatment with cercosporamide was initiated 24h after injection of the B16 cells, after the initial colonization of the lungs; these experiments could not have assessed the potential of preventing invasion by blocking eIF4E phosphorylation. Therefore, MNK inhibitors may possess important unexplored properties as metastasis preventive agents, which we will touch upon in Chapter 3. This issue will likely be addressed with the development of novel MNK inhibitors by Bayer and eFFECTOR, which are currently in clinical trials.

There are currently several inhibitors in clinical trials targeting eIF4E expression via antisense oligonucleotides, its availability through mTOR inactivation or its recruitment to the eIF4F complex by blocking the eIF4E-eIF4G interaction (319). However, the application of some of these inhibitors, as well as currently used therapeutic agents such as rapamycin (299), gemcitabine (298), cytarabine (425) and herceptin (118), leads to increased eIF4E phosphorylation and concomitant resistance to treatment. Therefore, inhibitors of eIF4E phosphorylation may be useful in preventing resistance to some anti-cancer drugs, in addition to blocking metastatic progression. Confirming this idea, in the years since the experiments in Chapter 2 were published, several reports highlighted the propensity of MNK inhibition to sensitize cancer cells to chemotherapy, radiotherapy and targeted therapy (232-234, 426). The promise of MNK inhibitors to provide clinical benefits is growing, and will be further discussed in Chapters 3 and 4.

2.4 MATERIALS AND METHODS Reagents: TGFβ was purchased from PeproTech (USA). Insulin was from Sigma. Scrambled control siRNA and MNK1-specific siRNAs were from IDT (USA).

Mice and tissue preparation: eIF4ES209A mice (55) were bred with MMTV-PyMT (412). Tumor onset in the mammary glands of control and experimental virgin mice was determined by palpation 3 times per week. Tumor growth was monitored by caliper measurements once per week or every 2 days near the experimental end point (single tumor above 2 cm3 or total burden above 6 cm3), at which point lung and tumor samples were collected. Step sections covering the entire lung were obtained and metastases were counted on all slides. The slide with the highest number of metastases was considered representative and used in further analyses. Lung and tumor samples were fixed in 10% formalin for paraffin imbedding and sectioning at 6 μm thickness. Pulmonary metastases were counted on H&E-stained 50 μm step- sections. Serial sections of tumor samples were obtained for immunohistochemistry. Immunohistochemistry was performed using the Vectastain ABC peroxidase system (Vector Labs, USA). Stained sections were quantified using Aperio ImageScope software using the positive pixel count algorithm. All experiments involving animals were conducted in accordance with McGill University (Montreal, QC, Canada) animal care guidelines.

Cell lines: 67NR, 168FARN and 4T07 cells were obtained from Fred Miller (Karmanos Institute, Detroit, USA). Cells (5x105) were injected into each 2nd mammary fat pad of BALB/c mice. Thirty days later, mice were sacrificed and the primary tumor harvested. Tumors obtained were immediately snap-frozen and pulverized under liquid nitrogen and stored at -80oC until further use. Extracts were prepared by suspending the specimens in RIPA lysis buffer (427) and used for immunoblotting. Wild-type and eIF4ES209A transformed MEFs were obtained as described (55). For rescue experiments, MEFs were transfected with pcDNA3.1 constructs using lipofectamine 2000 and selected with G418 sulfate. Normal Murine Mammary Gland (NMuMG) epithelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%

FBS, insulin, 2mM glutamine, 100 U/ml penicillin, and streptomycin. MCF10A cells were cultured in DMEM supplemented with 5% horse serum, hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), epidermal growth factor (20 ng/ml), and penicillin-streptomycin (100 μg/ml each). NMuMG-BMN, NMuMG-HA-MKK3AL, and NMuMG-Flag-p38AGF cells were kindly provided by A. Bakin (Roswell Park Cancer Institute) and cultured in the same media as parental NMuMG cells. MCF10A stable cells expressing eIF4E or eIF4ES209A were infected with retrovirus produced in phoenix 293T cells, and subsequently selected with G418 sulfate and propagated. Cells were subsequently infected with lentiviral particles containing non-target control shRNA or human eIF4E shRNA (Sigma, USA). siRNAs were introduced into NMuMG using RNAiMAX (Life Technologies, USA) in accordance with the manufacturer's protocol.

Western blot analysis: Cell monolayers were washed with PBS and harvested with trypsin. Washed cells were pelleted and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Roche). The lysate was cleared by centrifugation and protein concentration was determined with the Bio-Rad protein concentration assay solution (Bio-Rad, Mississauga, ON). 25 to 150 micrograms of protein were separated by 10-12% SDS-PAGE and electroblotted onto a nitrocellulose membrane. Membranes were incubated overnight at 4°C with the indicated primary antibodies, and the following day incubated with the appropriate secondary anti-rabbit or anti-mouse antibodies for 1-3h at room temperature. Membranes were developed with the enhanced chemiluminescence Western Blot Detection Kit. All Western blots shown are representative of at least 3 independent experiments.

Fluorescence microscopy: Cells were cultured on glass coverslips in 12-well plates for 24h, then fixed with ethanol:acetic acid at -20oC, incubated with antibodies against the epithelial marker ZO-1 and then incubated with Alexa fluor 488 conjugated goat anti-rabbit IgG (Life Technologies, USA). Nuclei were stained with Hoechst. The mounted samples were scanned with a Leica DM LB2 microscope. Differences in ZO1 immunofluorescence were quantified by selecting a defined area corresponding to edges between two cells and by calculating the imageJ parameter “RawIntDen”, which is the sum of the pixel values in the selected area.

Migration and invasion: Random migration was monitored by time-lapse microscopy and analysed using MetaMorph Automation & Image Analysis Software (Molecular Devices, USA); 30,000 cells were plated in matrigel-coated 6 well plates and stimulated with 10% serum. Transwell migration assays were performed using 24-well cell culture inserts (BD Biosciences, USA) as described (428). Briefly, 30,000 cells were plated in the inserts in DMEM with 0.5% serum, which were placed in wells containing DMEM with 10% serum. After 16 hours, cells were counted on the bottom side of the inserts. Invasion index was obtained as the percentage of transwell migration with/without coating of the inserts with 50μL 5% growth factor-reduced (GFR) matrigel (BD Biosciences, USA). For colony outgrowth assays, 2000 cells were plated into 50μL GFR matrigel in 96-well plates and incubated for 7 days.

Plasmids and constructs: eIF4E and eIF4ES209A constructs were described (55, 98). pcDNA3.1- FLAG-eIF4EWT was described (275) and used to obtain pcDNA3.1-FLAG-eIF4ES209A by quick- change PCR mutagenesis with the following primers: 5’-GACACAGCTACTAAGGCAGGCTCCACCACTAAAAATAGGTTTGTTGTTTAAGAAG-3’, 5’-GTGGTGGAGCCTGCCTTAGTAGCTGTGTCTGCGTGGGACTGATAACC-3’. MMP-3 cDNA was obtained from Origene (USA) in pCMV6-XL4 and cloned into pcDNA3.1 using the NotI restriction sites. pCDNA3.1-HA-SNAIL was obtained from Addgene (USA).

Polysome profile analysis, RNA isolation and RT-qPCR: Polysome profile analysis was carried out as described (275). RNA from each fraction was isolated using easy-BLUE kit (FroggaBio, Canada) and treated with DNaseTurbo (Ambion, USA) according to the manufacturer's instructions. Reverse transcription PCR (RT-PCR) of 100ng RNA of each fraction was carried out using SuperScript III First-Strand Synthesis System (Invitrogen). qPCRs were carried out in a Mastercycler Realplex2 (Eppendorf) system using iQ Sybr green Supermix (Bio-Rad) according to the manufacturer's instructions.

Bioinformatics analysis: To identify genes whose translation is sensitive to eIF4E- phosphorylation we reanalyzed a previously generated microarray data set, obtained by polysome profile analysis of WT and eIF4ES209A MEFs (55). To enable analysis of differential translation using the anota algorithm (429), we simulated a third replicate as described (430). This analysis results in an increase in false positives, but provides a ranking of genes that can be used for robust analysis, such as analysis of overrepresentation of genes belonging to specific pathways. To identify differential translation we used anota with the following settings: maxSlope=1.5, minSlope=(-0.5), maxRvmPAdj=0.15, selDeltaPT=log2(1.5) and minEff=log2(1.5). Identified differentially translated genes were further analyzed using DAVID (431).

2.5 ACKNOWLEDGMENTS This work was by supported by The Susan G. Komen Breast Cancer Foundation (IIR12224057), and the Canadian Cancer Society (2010-700377) to N.S. W.H.M. was supported by the Cancer Research Society (2012-17280). We thank I. Topisirovic for advice; C. Zakaria, A. Sylvestre, S. Perreault and C. Lister for technical assistance, N. Siddiqui for critical reading of the manuscript and S. Ramón y Cajal (Vall d'Hebron University Hospital, Barcelona, Spain) for his support and insights into tumor heterogeneity. We thank the Animal Facility and the Histology Facility at the Goodman Cancer Research Centre for mouse work and tissue processing.

CHAPTER 3

TRANSLATIONAL CONTROL IN THE TUMOR MICROENVIRONMENT PROMOTES LUNG METASTASIS VIA PHOSPHORYLATION OF EIF4E IN NEUTROPHILS

3.1 CONTEXTUAL SETTING While our understanding of how translation is deregulated in cancer cells is growing, we know very little about translational control in cells that comprise the tumor microenvironment (TME). Yet, tumors are composed of many heterogeneous cell types, including endothelial cells, fibroblasts, adaptive and innate immune cells, which comprise the bulk of some tumors. Stromal cells play important roles in all steps of tumor development, growth, vascularization and metastasis (432). They also must respond to similar stresses and challenges as cancer cells (433), and purportedly can achieve this by regulating protein synthesis, though data explicitly demonstrating this is lacking. It is thus imperative to understand whether modulating translation in cells of the TME affects cancer progression, especially considering the numerous anti-cancer agents targeting translation that are currently in clinical trials (102). When initiating this project, the possibility that translation in cells of the TME could play important roles in cancer had been indirectly suggested by a variety of studies demonstrating the importance of translation in non-transformed cell types. For example, translation plays important roles in the nervous system, where both eIF4F and eIF2 regulate aspects of behavior, including learning and memory, depression, autistic-like social and obsessive behaviors, as well as pain sensing (434-438). Translational control, particularly by the MNKs, of cytokine production in a variety of immune cell types had also been described in models such as Natural Killer T cell-mediated liver damage, Toll-like receptor activation in macrophages, neutrophil activation by LPS, interferon production and cellular responses to interferon during viral infections (381-385, 439-441). In T cells, phosphorylation of eIF4E had been linked to autoimmune encephalomyelitis, T helper type 17 differentiation in multiple sclerosis, whereas limiting eIF4E expression controls the differentiation of T regulatory cells (385, 442). Thus, the function of many cell types can be regulated by translation; however, whether this could have a significant impact on any aspect of cancer had never been demonstrated.

Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression by investigating the importance of eIF4E phosphorylation in the TME of breast cancer patients, as well as by utilizing the eIF4ES209A mouse model. By orthotopically

77 injecting a syngeneic mammary tumor cell line, 66cl4, in wild-type (WT) and eIF4ES209A mice, we show that translational control in the TME plays a crucial role in metastatic progression, at least in part by modulating the survival of pro-metastatic neutrophils, which can be targeted using the small molecule merestinib.

3.2 RESULTS 3.2.1 Phosphorylation of eIF4E in breast cancer patients We previously demonstrated that eIF4E phosphorylation in cancer cells promotes metastatic dissemination of mouse mammary tumors (97). To assess its importance in patients, we performed immunohistochemical analysis of eIF4E phosphorylation in human breast cancer samples in a tissue microarray representing tumors from 56 patients with associated normal tissue. Of the 56, 40 samples were of sufficient quality for analysis, and the expression of eIF4E phosphorylation was scored as high or low (see scoring examples in Figure S3.1). Interestingly, phosphorylated eIF4E was readily detectable in the stroma of patient samples, in addition to the neoplastic cells (Figure 3.1A); samples were therefore also separated based on the presence or absence of phospho-eIF4E in the TME. Phosphorylation of eIF4E was more common in both the cancer cells and TME of tumor tissue than in associated normal samples (Figure 3.1B-C). No differences were observed in the survival of patients when stratified according to eIF4E phosphorylation in the cancer cells (Figure 3.1D). Previous reports have indicated that eIF4E phosphorylation may be associated with survival in some cancer types but not others (115, 356, 387, 424, 443), which could be explained by the dominant role of deregulated mTOR/4E-BP/eIF4E axis (116, 282). In striking contrast, none of the patients in which eIF4E phosphorylation was absent in the TME succumbed to their disease (Figure 3.1E). This group also tended to present tumors of lower grade, possess a negative lymph node status, express at least one targetable hormone receptor, and be present in older patients (Figure S3.1). This may reflect a more prominent role of the MNKs in non-transformed cells. However, the sample size available for this experiment was too small to be conclusive, despite the statistical significance of the result (Figure 3.1E). Notwithstanding the limitations, our data suggest that eIF4E phosphorylation in the TME is common in breast cancer patients and is associated with poor survival, prompting us to investigate this phenomenon in a mouse model.

Figure 3.1. Phosphorylated eIF4E in the TME of breast cancer patients correlates with poor survival. (A) Representative image of a TMA core displaying immunohistochemical detection of phospho-eIF4E in cancer cells (red arrow) and cells of the surrounding stroma (red brace) in a DCIS sample from a patient with triple negative breast cancer. (B) Distribution of samples displaying high or low eIF4E phosphorylation in the epithelial cells of normal and malignant mammary tissue. (C) presence or absence of phospho-eIF4E in the TME of normal and malignant mammary tissue. (D-E) Overall survival of breast cancer patients stratified according to eIF4E phosphorylation in the epithelial cells (D) or TME (E).

3.2.2 Phosphorylation of eIF4E in the TME promotes metastasis in vivo To explore the role of translational control in the TME, we utilized the 66cl4 syngeneic mouse mammary tumor model. This cell line was chosen based on an interesting paradox in its metastatic properties: while it is well known to be incapable of invading blood vessels in a chorioallantoic membrane (CAM) assay, it metastasizes to the lungs when implanted in the fat pad of a syngeneic (BALB/c) host (411). As the metastases form in the lung parenchyma rather than within capillaries, the tumor microenvironment enables extravasation and metastasis in this model. When WT and eIF4ES209A mice were subjected to orthotopic injections with 66cl4 cells, primary tumors developed with similar onset and growth rates in both mouse strains (Figure 3.2A-B). Consistent with these observations, all tumors were histologically indistinguishable, and immunohistochemical analysis revealed no differences in proliferation, apoptosis or vascularization (Figure 3.2C, Figure S3.2A). However, the absence of eIF4E phosphorylation in the TME of eIF4ES209A mice was associated with a nearly complete resistance to the development of lung metastases (Figure 3.2D-F). In stark contrast, WT animals averaged 25 metastases/lung and displayed metastatic burdens of up to 50% of the total lung area. Interestingly, the few metastases present in eIF4ES209A mice tended to be intravascular, suggesting a possible barrier to extravasation in these mice (Figure 3.2E-F). Similar results were obtained using a different model, in which EO771 cells were injected in C57/BL6 mice bearing the eIF4ES209A mutation or lacking the MNKs (Figure S3.2B). Thus, the ability of cells within the TME to phosphorylate eIF4E has a significant impact on metastasis.

Figure 3.2. Phosphorylation of eIF4E in the TME promotes lung metastasis but has no effect on primary tumor growth. Onset (A) and growth (B) of tumors derived from 66cl4 cells injected into the mammary fat pad of WT and eIF4ES209A mice (n=10). Tumors were assessed for differences in histology (H&E) and in the expression of the indicated markers by immunohistochemistry (C). Quantification (D) and representative image (F) of the number of lung metastases in tumor-bearing mice (n=16). Quantification (E) and representative examples (F, lower panel) of the proportion of intravascular lung metastases.

3.2.3 Excluding potential mediators of the effect of phospho-eIF4E in the TME To identify cell type(s) with phospho-eIF4E-dependent effects on metastasis, immune profiling was performed in the spleen, bone marrow and lymph nodes of non-tumor bearing WT and eIF4ES209A mice. No differences were observed in the steady-state levels of any of the cell types analyzed, including monocytes, granulocytes, T cell subpopulations and B cells, or in cytokine production by T cells (Figure S3.2C-G), indicating that phosphorylation of eIF4E is dispensable for normal hematopoiesis. In the context of 66cl4 mammary tumors, previous studies have found neutrophils, natural killer (NK) cells and the ratio of CD8+ T-cells to T-regulatory

+ (CD8 /Treg) cells to be related to metastatic potential (444-446). However, analysis of

+ dissociated primary mammary tumors indicated that the CD8 /Treg ratio remained unchanged in WT and eIF4ES209A mice (Figure S3.3A). To further investigate the role of T cells, we compared WT fully immune competent BALB/c mice to T-cell deficient BALB/SCID mice. Tumor growth and metastatic burden were similar in both strains (Figure S3.3B, C), indicating that T cells are functionally suppressed in this model. Taken together, these data indicate that T cells are unlikely to mediate the effect of eIF4E phosphorylation in the TME. To explore the role of NK cells in our model, we performed lung colonization experiments by injecting 66cl4 cells in the tail vein of WT and eIF4ES209A mice following NK cell depletion. Interestingly, depletion of NK cells led to a three-fold increase in lung nodules, confirming the importance of this cell type as a first line of defense against metastatic colonization (Figure S3.3D). However, the observed increase was, if anything, greater in WT than eIF4ES209A mice, indicating that NK cell-mediated protection from metastasis was at least as effective in WT mice and could therefore not be responsible for the observed decrease in lung metastasis in eIF4ES209A mice. Strikingly, the difference in lung colonization between WT and eIF4ES209A mice, which was dramatic in mice bearing primary tumors, was negligible in the experimental metastasis assay, where cells were injected in the tail vein in the absence of a primary tumor (Figure S3.3D). This observation suggests the requirement for a pre-metastatic niche, the formation of which would be dependent on eIF4E phosphorylation within cells that comprise the lung microenvironment.

3.2.4 Neutrophils promote metastasis Neutrophils have been implicated in the formation of a pre-metastatic niche in the lungs of tumor bearing mice (447). We therefore wished to investigate the contribution of neutrophils to lung metastasis in eIF4ES209A mice. Splenomegaly was apparent in tumor-bearing WT mice, in which the spleens weighed nearly seven times more than those of non-tumor bearing mice due to a more than 100-fold increase in neutrophils (Figure 3.3A-C). This was mirrored in the blood of tumor bearing WT mice (Figure 3.3D), where neutrophils accumulate as early as two weeks post injection of 66cl4 cells in the mammary fat pad and reach concentrations averaging 150*109 cells/L by four weeks (Figure 3.3D); normal concentrations were in the range of 0.5- 3*109 cells/L (Figure 3.3D). Importantly, at the metastatic site, neutrophils represented <20% of cells in naïve mice, which increased to an average of 60% in animals with lung metastases (Figure 3.3E-F). This accumulation correlated with metastatic burden (Figure 3.3E). In striking contrast, neutrophil accumulation in eIF4ES209A tissues was minimal (Figure 3.3D-F). It is noteworthy that neutrophil infiltration in primary tumors was mostly absent, except in necrotic areas, in both backgrounds (Figure 3.3F), possibly explaining why phospho-eIF4E in the TME had little impact on primary tumor development (Figure 3.2A-B).

To establish a causative role for neutrophil accumulation in promoting metastasis in this model, we performed depletion experiments using antibodies specific to Ly6G (clone 1A8). This marker is expressed on myeloid cells in the bone marrow but is restricted to neutrophils in peripheral tissues, and its targeting with anti-Ly6G antibodies depletes neutrophils without affecting other white blood cells (Figure S3.3E-F). In our model, both circulating mature neutrophils and immature neutrophils (band cells) were successfully depleted in peripheral blood by anti-Ly6G treatment (Figure 3.4A-B). Neutrophil depletion conferred significant protection from lung metastasis, but had no effect on primary tumor growth (Figure 3.4C-D). Thus, neutrophil depletion recapitulated our findings in eIF4E phosphorylation deficient mice, supporting the conclusion that neutrophils mediate the different metastatic dispositions of WT and eIF4ES209A mice.

Figure 3.3. Neutrophils accumulate in tissues of tumor-bearing WT and eIF4ES209A mice. The spleens of naïve and tumor-bearing mice were isolated (A) to determine their weight (B) and cellular composition (C). Shown in purple are CD11b positive cells, identified as monocytes (top left quadrant) and granulocytes (bottom right quadrant) based on the expression of Ly6C and Ly6G. CD11b negative cells are shown in blue and are Ly6C/Ly6G negative (bottom left quadrant). (D) Circulating neutrophil concentrations in the blood of tumor bearing mice (n=4). (E-F) Quantification and representative images of neutrophils in lung and tumor sections based on immunohistochemical detection of Ly6G. (E) The percentage of Ly6G positive cells of Ly6G- stained lungs (F) was determined using an automated quantificiation algorithm in ImageScope and plotted against the number of lung metastases to determine their correlation (r=0.62 Spearman correlation). Ly6G was largely absent in primary tumors (F, right panel).

Figure 3.4. Anti-Ly6G depletion of neutrophils impairs metastasis. Neutrophils were depleted from WT and eIF4ES209A mice bearing palpable 66cl4 tumors (7 days post injection) by daily intra-peritoneal injections of an anti-Ly6G antibody (clone 1A8). Control mice received injections of a non-specific antibody of the same isotype (labelled “IgG”). Quantification of circulating neutrophils (A) and band cells (B) in naïve mice and in mice at four weeks post- injection of 66cl4 cells. Tumor growth was monitored throughout the experiment by caliper measurements (C). Lung metastases were detected by H&E staining and quantified in (D). N=4- 5 mice per condition.

3.2.5 Phospho-eIF4E promotes neutrophil survival and accumulation Many pro-metastatic roles have been described for neutrophils, including proteolytic remodelling of the extra-cellular matrix, increased angiogenesis, enhanced extravasation and immune suppression of multiple cell types including T cells and NK cells (reviewed in (447)). However, none of these activities, when assayed ex vivo, differed significantly in response to eIF4E phosphorylation (Figure S3.4A-C), highlighting the importance of eIF4E phosphorylation in neutrophil accumulation, rather than function, in our system. Furthermore, in tumor-bearing mice, circulating levels of cytokines and chemokines involved in neutrophil recruitment were similar in both genotypes (Figure 3.5A). In particular, G-CSF, which is tumor-derived in this model, increased 100-fold in tumor bearing WT and eIF4ES209A mice equally (Figure 3.5A).

Next, we explored the possibility that neutrophil differentiation and/or survival were dependent on eIF4E phosphorylation. Myeloid progenitor cells defined as Lin- Sca1+ cKit + (LSK) were isolated from the bone marrow of WT and eIF4ES209A mice and differentiated to neutrophils ex vivo in the presence of G-CSF (Figure S3.4D). Both WT and eIF4ES209A LSK cells were differentiated to neutrophils, but the numbers of eIF4ES209A neutrophils obtained was reduced due to decreased viability (Figure S3.4E-F). Strikingly, blood smears revealed a 6-fold increase in the proportion of apoptotic neutrophils in eIF4ES209A tumor-bearing mice, corresponding to fewer total circulating neutrophils (Figure 3.5B-C). Furthermore, neutrophils isolated from the blood of tumor-bearing eIF4ES209A mice displayed reduced G-CSF-induced survival when cultured ex vivo (Figure 3.5D). Interestingly, no differences were observed between WT and eIF4ES209A neutrophils when cultured in the absence of G-CSF (Figure 3.5D). This may explain why eIF4E phosphorylation didn’t affect the circulating neutrophil numbers of naïve mice.

Previous studies have established the key role of translation in the regulation of expression of members of the BCL2 family of anti-apoptotic proteins (113, 290, 448). Accordingly, analysis of circulating neutrophils isolated from the blood of tumor bearing mice revealed that eIF4ES209A neutrophils express lower amounts of the anti-apoptotic proteins BCL2 and MCL1

Figure 3.5. Phosphorylation of eIF4E promotes neutrophil survival in response to G-CSF. (A) Multiplex ELISA results quantifying the indicated cytokines and chemokines from the serum of naïve (left) (n=5) or tumor bearing (right) (n=9) WT and eIF4ES209A mice. (B-C) The survival of circulating WT and eIF4ES209A neutrophils was assessed on Giemsa-stained blood smears of tumor-bearing mice. (B) Total numbers of live and apoptotic neutrophils counted in a microscope field at 40X magnification (n=5). (C) Proportion of live and apoptotic neutrophils counted in A. (D) Apoptosis in response to cytokine withdrawal was assessed in circulating neutrophils from tumor-bearing mice: neutrophils were isolated by density gradient centrifugation and incubated for 24h in the presence or absence of G-CSF; apoptotic neutrophils were identified by flow cytometry, shown is percentage of Ly6G positive cells stained positive for Annexin V (n=3). (E) Neutrophils were isolated from the blood of tumor bearing mice by density gradient centrifugation and lysed for western blot analysis of the indicated proteins.

Figure 3.6. Merestinib inhibits eIF4E phosphorylation, neutrophil survival and metastatic progression of 66cl4 tumors. (A-C) BALB/c mice bearing palpable 66cl4 tumors were treated with 12mg/kg merestinib daily for 14 days. (A) Immunohistochemical analysis of primary tumors and lungs; (B) tumor growth; and (C) metastatic burden of vehicle and merestinib treated animals. (D) Circulating neutrophil concentrations in the blood of vehicle and merestinib-treated animals 14 days post-injection. (A-D) n=4 mice per group. (E) Neutrophils were isolated from the blood of 8 tumor-bearing mice and treated ex vivo with or without merestinib (100nM) for 24 hours in the presence of G-CSF; their survival was monitored by flow cytometry. (F-G) 66cl4 cells were treated with merestinib in vitro for the indicated times and concentrations; proliferation was monitored by trypan blue exclusion, and Western blot analysis of the indicated proteins was performed on lysates of 66cl4 cells treated for 4 hours (G).

Figure 3.7. MNK inhibition blocks eIF4E phosphorylation, neutrophil survival and metastatic progression of 66cl4 tumors. (A-C) BALB/c mice bearing palpable 66cl4 tumors were treated with a MNK inhibitor (MNKi) daily for 14 days. (A) Immunohistochemical analysis of primary tumors and lungs; (B) tumor growth; and (C) metastatic burden of vehicle and MNKi treated animals. (D) Circulating neutrophil concentrations in the blood of vehicle and MNKi-treated animals 28 days post-injection. (A-D) n=5 mice per group. (E) Neutrophils were isolated from the blood of 4 tumor-bearing mice and treated ex vivo with or without MNKi for 24 hours in the presence or absence of G-CSF; their survival was monitored by flow cytometry, shown is percentage of Ly6G positive cells positively stained with Annexin V.

(Figure 3.5E). This is consistent with a previous report linking neutrophil survival to regulation of MCL1 by MNKs (449). Thus, our results demonstrate that neutrophil accumulation is reduced in eIF4ES209A mice due to impaired resistance to apoptotic cell death.

3.2.6 Targeting eIF4E phosphorylation in the tumor and TME prevents metastasis Our results indicate that eIF4E phosphorylation in the TME promotes metastasis by increasing neutrophil survival. Thus, small molecule inhibitors of eIF4E phosphorylation would be expected to prevent metastatic progression, especially considering our previous work demonstrating that eIF4E phosphorylation in cancer cells promotes metastasis. To investigate this, eIF4E phosphorylation was blocked in vivo using merestinib, a multi-kinase inhibitor that was recently demonstrated to be a potent MNK inhibitor reducing the translation of Bcl2 and Mcl1 in mouse xenograft and allograft models (365, 450-452). In our model, merestinib inhibited eIF4E phosphorylation in the primary tumor and metastatic site (Figure 3.6A). This treatment had no effect on primary tumor growth, but prevented neutrophil accumulation and the development of lung metastases (Figure 3.6B-D). Furthermore, a 100nM concentration of merestinib was sufficient to inhibit the survival of neutrophils cultured ex vivo in the presence of G-CSF (Figure 3.6E). In contrast, there was no effect on the proliferation of 66cl4 cells treated with this dose of merestinib (Figure 3.6F), despite efficient inhibition of eIF4E phosphorylation starting at 10nM (Figure 3.6G), which is consistent with the finding that merestinib had no effect on the growth of primary tumors (Figure 3.6A). Importantly, merestinib did not inhibit mTOR signaling, as phosphorylation of 4E-BP1 and S6 were unaffected (Figure 3.6G), whereas MAPK signaling was only affected at higher doses that correlated with decreased proliferation, as shown by ERK dephosphorylation at 1μM and 10μM merestinib (Figure 3.6G). To validate these findings, a second MNK inhibitor was used. This inhibitor is reported to be highly specific for the MNKs, but Material Transfer Agreements prevent us from disclosing its identity. Despite this limitation, MNK inhibition confirmed our findings using merestinib, as eIF4E phosphorylation decreased in tumors and lungs (Figure 3.7A), which had no effect on primary tumor growth (Figure 3.7B) but was accompanied by a reduction in circulating neutrophils (Figure 3.7C) and lung metastasis (Figure 3.7D). Furthermore, MNK inhibition blocked

97 neutrophil survival in response to G-CSF (Figure 3.7E). Taken together, these results support our findings that eIF4E phosphorylation promotes metastatic progression via increased neutrophil survival, and argue for its chemical inhibition in cancer patients using MNK inhibitors such as merestinib.

3.3 DISCUSSION The experiments documented herein establish for the first time an important role for translational control in cells of the TME. Using an orthotopic model wherein 66cl4 mammary tumor cells are injected into syngeneic BALB/c mice, we demonstrate that eIF4E phosphorylation in the non-transformed cells of the host promotes metastatic lung colonization. We validate this finding in an unrelated orthotopic model using EO771 cells and syngeneic C57/BL6 mice. We show that eIF4E phosphorylation promotes neutrophil survival, thus favoring metastasis to the lung. Interestingly, we demonstrate that eIF4E phosphorylation in the TME of breast cancer patients correlates with poor outcome, though validation studies with larger sample sizes are critical to confirm this effect.

Our data clearly implicate neutrophils in the development of lung metastases from 66cl4 tumors. Emerging evidence suggests that neutrophils represent a phenotypically diverse cell population, with subsets possessing pro-metastatic versus anti-metastatic functions (445, 447, 453-455). In our model, pro-metastatic functions clearly dominate. Neutrophil depletion reduces lung colonization in WT mice, and fails to enhance colonization in eIF4ES209A mice, which would be expected if anti-metastatic neutrophils were playing a major role. This contrasts the conclusions of Granot et al., who report that neutrophil depletion increases lung metastasis (454). However, most of the experiments in the latter publication use the related 4T1 cells, which elicit differential neutrophil responses. In one experiment, circulating neutrophil numbers in mice bearing 66cl4 tumors were much lower than in mice bearing 4T1 tumors, and in fact similar to those seen in naïve mice, in contrast to our results (454). This distinction might arise simply from a difference in the time of analysis: Granot et al. measure neutrophil levels 7 days after engraftment. At this time point, 66cl4 tumors are barely palpable,

98 circulating G-CSF levels are low and neutrophils have yet to accumulate in the circulation, whereas 4T1 tumors grow much faster and produce more G-CSF than 66cl4 tumors. In our experiments with 66cl4-derived tumors, neutrophils accumulate by day 14 and reach peak concentrations around 150*109/L after 3-4 weeks (Figure 3.4). Thus, our results are consistent with previous findings showing that neutrophils favor the metastatic process (445, 455).

Our experiments did not identify any specific neutrophil functions affected by eIF4E phosphorylation, as none of those tested changed significantly between WT and S209A neutrophils. As these experiments were by no means exhaustive, there may be additional layers of phospho-eIF4E mediated regulation of neutrophils that we were unable to identify (456). It is also possible that metastatic cancer cells seeded in the lungs attract neutrophils. However, neutrophil infiltration to the lung occurs as early as 2 weeks following primary tumor injection, mirroring their accumulation in the blood; this is earlier than the appearance of macroscopic nodules (data not shown). In addition, neutrophil infiltration was limited in the primary tumors, but extensive in the blood, spleen and lungs (Fig. 3.3). It seems unlikely that such a tissue distribution would occur if cancer cells were facilitating neutrophil recruitment to the lungs, rather than the other way around. Our data therefore supports the notion that aberrant neutrophil accumulation is a cause, rather than an effect, of metastasis to the lungs.

Unexpectedly, our results demonstrate that apoptosis in neutrophils is regulated by the phosphorylation of eIF4E, while no such effect is seen in 66cl4 cells, MEFs or in primary tumors (Figure 3.5) (55, 97). In this regard, it is noteworthy that immature neutrophils express BCL2, whereas mature neutrophils depend primarily on MCL1 for survival, in contrast to other cell types that may express a battery of anti-apoptotic proteins (457). As they seem to rely on individual BCL2-family members at any given time, neutrophils may be particularly sensitive to translational regulation of BCL2 and MCL1. Several groups have reported the regulation of these factors by eIF4E levels and phosphorylation (113, 448). The impact of eIF4E phosphorylation on apoptosis is therefore highly context specific. This may explain why our previous studies using prostate and breast cancer models found no link between apoptosis and

99 eIF4E phosphorylation, in contrast to other groups studying models of lymphoma and melanoma (113, 290).

Several other cell types may be playing important roles that we did not identify in our mouse model. Possibilities include macrophages, in which eIF4E phosphorylation has been linked to cytokine production (381, 458), as well as regulatory T cells, where eIF4E levels have been reported to play important roles (380), and IFNγ production by Th1 cells, which has recently been linked to the p38-MNK-eIF4E axis (383). The case of T cells is interesting, as our data indicate that they are functionally suppressed by neutrophils in the WT setting (Figure S3.3A-C, 4B), but do not preclude a role for phospho-eIF4E in these cells when relieved from neutrophil suppression in the eIF4ES209A setting. In fact, the translational regulation of various cytokines by these cells reported in other contexts (383, 458), as well as data presented by the pharmaceutical company eFFECTOR regarding the effects of their own MNK inhibitors (459), support this notion. In contrast, work with T cells lacking MNKs has indicated that these kinases, and eIF4E phosphorylation, are dispensable for normal T cell function, but indirectly affect their differentiation and activity in a T cell extrinsic manner, using an in vivo model of autoimmune encephalomyelitis (385, 458), mirroring our own findings in the TME.

Merestinib had no effect on primary tumor growth in our model, despite inhibiting tumor growth in mouse models of non-small cell lung cancer, leukemia and glioblastoma (365, 451, 452, 460). As discussed above, this may be reflective of the importance of phospho-eIF4E sensitive anti-apoptotic factors in various cell types. However, it is also likely that these differences arise from the lack of specificity of merestinib. Indeed, this inhibitor targets an array of kinases including MET, for which it was originally developed (452). Any of these other targets may contribute to the effects of merestinib observed by us and others, and their relative expression and importance in these various models has not been compared. Interestingly, our in vitro assays demonstrated that merestinib inhibits eIF4E phosphorylation at doses much lower than those required to inhibit proliferation (Figure 3.6F-G). It is therefore likely that targets other than the MNKs mediate the anti-proliferative effects of merestinib, that their

100 inhibition in 66cl4 cells require higher doses of the drug, and that such high doses were not reached in our in vivo model. Whether anti-proliferative doses of merestinib can be achieved in patients will need to be determined. Of note, the similar effects obtained when using merestinib or a more specific MNK inhibitor were similar, arguing for primarily MNK-specific effects of merestinib in our model. Irrespective of these issues, we and others show that merestinib treatment has clear beneficial properties in cancer cells and in the TME, supporting its clinical development.

Possible clinical implications of our findings are noteworthy. Approximately 10% of cancer patients present with pathologically high levels of neutrophils (>20-50*109 cells/L of blood) (461). This can be caused by high levels of G-CSF expression from primary tumors and is linked to lung colonization and poor prognosis (461). However, the interplay between cancer and neutrophils is complex, as the main concern for most patients is neutropenia resulting from chemotherapy, where insufficient neutrophils lead to an inability to efficiently combat bacterial infections. These patients are generally treated with G-CSF to stimulate neutrophil production by the bone marrow (461). Thus, depleting neutrophils with monoclonal antibodies in the manner used in this and other publications is not practical in the clinic. However, inhibiting eIF4E phosphorylation, which prevents pathological accumulation of neutrophils in our mouse model, but not normal neutrophil homeostasis, may be beneficial to cancer patients. This is an especially attractive approach, as eIF4E phosphorylation appears to be dispensable for normal immune cell development (Figure S3.2). In addition, our previous findings indicate that eIF4E phosphorylation in cancer cells also promotes metastatic progression, further arguing for the development of MNK inhibitors for the prevention of metastasis (97, 290). However, as eIF4E phosphorylation has no effect on primary tumor growth (Figure 3.2A-B), such a phospho-eIF4E targeting therapy would most likely be useful in combination with other treatments.

Immunotherapeutic drugs, including inhibitors of CTLA4 and PD1/PDL1, are particularly promising candidates to combine with MNK inhibitors. These compounds disrupt signals that suppress T cell mobilization, proliferation and function, leading to an increase in activated

101 tumor reactive T cells. The introduction of checkpoint inhibitors has led to a major revolution in the treatment of some cancer types such as melanoma (462). We and others have shown that neutrophils can inhibit T cell proliferation and function (Figure S3.4) (447), raising the possibility that preventing aberrant neutrophil accumulation with MNK inhibitors may improve the efficacy of immunotherapy. This is especially interesting considering the requirement for eIF4E phosphorylation in both the cancer and stromal compartments for efficient metastasis. In this regard, NF1 mutant melanomas may be particularly sensitive: they are expected to be responsive to immunotherapy due to their high mutational load (463), and sensitive to the effects of MNK inhibition similarly to other NF1 mutant tumors (464). This is a striking contrast to the immunosuppressive effects of many chemotherapeutic regimens and some targeted therapies such as rapamycin. Research in this direction is therefore highly warranted, and is promisingly expected to enter clinical trials later this year.

3.4 MATERIALS AND METHODS Experimental model and subject details Human samples. Human breast cancer samples were collected from needle biopsies of primary tumors from female patients between 30 and 90 years of age (see Figure S3.1) undergoing treatment at the McGill University Health Centre (MUHC) between 1999 and 2004 who provided written, informed consent (MUHC REB protocols SDR-99-780). Information regarding clinical variables was obtained through review of medical records. Haematoxylin and eosin (H&E)-stained sections from each sample were evaluated by an attending clinical pathologist with expertise in breast tissue to identify areas of normal tissue, DCIS and invasive carcinoma. Sections from biopsy cores were arranged as a tissue microarray on a single microscope slide used for immunohistochemistry. The TMA included samples from 56 patients, 40 of which were used for analysis. Reasons for excluding 16 patient samples were absence, or insufficient amount, of material on slide (12 patients), or tissue originating from a recurrence rather than a primary tumor (4 patients). Animal models. Immunocompetent WT BALB/c and eIF4ES209A mice (55) were bred in house after 12 backcrosses. SCID mice were from Jackson Laboratories. All experiments

102 involving animals were conducted in accordance with McGill University (Montreal, QC, Canada) animal care guidelines. Mice were maintained at 22oC with a continuous water supply, standard solid food and bedding, and a 12-hour light/dark cycle (light 6:30am-6:30pm). Treatment naïve were injected with 66cl4 cells between the ages of 8-12 weeks and weighed between 19 and 22 grams. Mice were examined twice weekly during tumor growth for caliper measurements and to monitor health and assess ethical endpoints (tumor size greater than 2cm3, dehydration, hunched back, weight loss), or daily for experiments with drug treatments or immune cell depletion. Tumor volume was estimated at the indicated time points using the formula V=(4/3*(3.14159)*(Length/2)*(Width/2)^2) and caliper measurements of the longest and shortest diameters of the tumor. Age-matched WT and eIF4ES209A mice were distributed in blocks of 2-3 mice of each genotype in cages of 4-5 mice. This blocking strategy was also used for merestinib treatments and neutrophil depletion. Experiments were repeated up to include the numbers of mice indicated in each figure legend (4-10 mice per group). Merestinib was provided by Eli Lilly and injected orally at 12mg/kg daily, formulated in 20% PEG 400/80% (20%

Captisol in H2O). For depletions, antibodies were injected intra-peritoneally with 50µL of anti- asialo-GM1 (eBioscience) for NK cells or 5.5mg/kg of anti-Ly6G (BioXCell) in saline for neutrophils. At endpoint, mice were anaesthetized with isoflurane, and then euthanized with

CO2 and cervical dislocation. For experiments requiring blood collection, heart punctures were performed following isoflurane anaesthesia, followed by cervical dislocation. Tissues were collected and processed immediately using 48-hour formalin fixation of tissues for immunohistochemistry. For experiments on neutrophil accumulation in circulation, 50μL of blood was collected weekly from the saphenous vein into EDTA coated capillary tubes. Cell culture. 66cl4 cells were obtained from W.H.M. These cells were originally isolated from a primary mammary tumor from a female BALB/c mouse due to a Mouse Mammary Tumor Virus infection. They were subsequently selected for resistance to thioguanine and ouabain. 66cl4 cells were cultured in DMEM (Wisent) with 10% fetal bovine serum (FBS)

o (Wisent) and 1% penicillin/streptomycin (P/S) (Sigma), at 37 C with 5% CO2. Neutrophils were isolated from the blood of tumor bearing mice by diluting 200μL in 5mL of PBS, which was loaded onto a Histopaque gradient consisting of a dense fraction of 3mL Histopaque 1119 and a

103 light fraction of Histopaque 1077. Columns were centrifuged at 800g for 30 minutes at room temperature and neutrophils were collected from the interface between the two Histopaque solutions, diluted in 10mL PBS and pelleted by centrifugation for 5 minutes at 800g. Residual RBCs were lysed by resuspending pellet in 5mL ACK buffer and incubating 5 minutes at room temperature. Neutrophils were then washed in PBS and resuspended in RPMI with 2% FBS and 1% P/S for ex vivo apoptosis experiments, or in PBS with 2% FBS for flow cytometry. To obtain high density and low density neutrophils, cells from the Histopaque interface and between the Histopaque 1077 and PBS layers were collected and further purified by flow cytometry using a BD Aria Fusion sorter.

Method details Immunohistochemistry. Formalin fixed and paraffin embedded samples from breast cancer patients and mouse organs were rehydrated by progressive washes for 5 minutes each in xylene (3x), 100% ethanol (2x), 70% ethanol, water. Antigens were retrieved using a citrate buffer pH 6 and heated for 15 minutes in a pressure cooker. Sections were washed in PBS, blocked in 2.5% normal horse serum for 10 minutes, followed by incubation with primary antibodies overnight. After PBS washes, endogenous peroxidases were quenched with 3% H2O2 in PBS for 5 minutes, followed by further PBS washes and incubation with HRP-conjugated ImmPRESS secondary antibodies. DAB was used as a peroxidase substrate for sample staining, Hematoxylin QS as a couterstain. All reagents except primary antibodies were from Vector Labs, primary antibodies used were anti-CD31 (Dianova), anti-phospho-eIF4E clone EP2151Y (Abcam), anti-ki67 (BD Pharmingen), anti-cleaved caspase 3 clone 5A1E (Cell Signaling), anti-Ly6G clone 1A8 (BD Pharmingen). In mouse lungs and tumors, percentages of cells positively stained with anti-Ly6G, ki67, CD31 or cleaved caspase 3 were determined using Aperio ImageScope software. Scoring of TMA for eIF4E phosphorylation in the epithelial cells and TME was performed independently by 2 blinded scorers. Phospho-eIF4E expression in cancer cells was categorized as “low” if undetectable or if expression was low and sparse; it was categorized as “high” if expression was uniformly detectable or if expression was sparse but very high. Phospho-eIF4E expression in the TME was categorized as “positive” if at least 5 cells outside the

104 tumor bed expressed phospho-eIF4E, otherwise it was categorized as “negative”. This number was chosen because it was small enough to allow analysis of sections with limited areas of stroma, but large enough to reduce the odds of a single randomly stained area affecting the analysis. The 2x2 scoring method was chosen to limit groups, due to the small sample number. Histology. Primary tumor histology as analysed in H&E stained sections by a pathologist with experience in breast cancer. Lung metastases were counted in 5 H&E stained step sections with 50μm spacing, metastases occurring in the same position of sequential sections were only counted once. Metastases were considered intravascular if entirely enclosed by an endothelial layer. All analyses were performed blind. Flow cytometery. For flow cytometric analysis, spleens were dissociated by manual crushing with a syringe plunger and cell strainer, bone marrow cells were washed out of mouse femurs with PBS using a 26-gauge needle after cutting off the extremities, tumors were dissociated by incubating minced pieces in collagenase B for 30 minutes at 37oC. RBCs were lysed by incubating in ACK buffer for 5 minutes. The resulting cell populations were washed in PBS, and 106 cells were resuspended in 100μL PBS with 2% FBS and various combinations of the following antibodies from BioLegend: APC-Annexin V, PE-anti-Ly6G, PerCP/Cy5.5-anti-Ly6C, APC/Cy7-anti-CD11b, AF488-anti-Sca1, PE-anti-cKit, AF700-anti-mouse lineage cocktail. Multiplex ELISA. Circulating cytokines were measured in the serum using Bio-Plex Pro Mouse Cytokine 23-plex Assay #M60009RDPD (Bio-Rad) following the manufacturer’s instructions. Serum was diluted 1:4 in sample diluent, and standards were prepared as recommended. Samples and standards were incubated in a 96 well plate with magnetic antibody-conjugated beads for 30 minutes at room temperature, followed by incubation for 30 minutes with the detection antibody and incubation with streptavidin-PE for 10 minutes at room temperature. Wash steps were performed between all incubations. Finally, samples were resuspended in Assay buffer and inserted into a Luminex 200 plate reader system. Cytokines below the detection limit of the assay were removed from further analysis. Cytokine withdrawal. Neutrophils isolated as described above were incubated for 24 hours in the presence or absence of 25ng/mL recombinant G-CSF (Peprotech) and 100nM merestinib at a density of 106 cells per well in a 12-well culture dish with 1mL culture medium.

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They were then collected by centrifugation at 800g for 5 min, washed in Annexin V staining buffer and resuspended in 100μL Annexin V staining buffer containing Annexin V and anti-Ly6G- PE for flow cytometric analysis. Proliferation. To monitor cell proliferation in response to merestinib, 106 66cl4 cells were plated in each well of 6 well plates. Individual plates were prepared for each of the 4 days included in the experiment, and the experiment was performed in triplicate. In all plates, every day following initial plating, media was replaced with fresh media containing vehicle (DMSO) or merestinib at the indicated concentrations. Every 24 hours of treatment, cells were trypsinized and counted in a set of 3 plates by trypan blue exclusion. Western blots. For merestinib treatments of 66cl4 cells, 5*106 cells were plated in each well of a 6-well plate, the following day, they were incubated with the indicated concentrations of merestinib for 4 hours and lysed in RIPA buffer. Protein concentration was determined using Bio-Rad Protein Assay and 20μg of protein was loaded on a 10% SDS-PA gel, then transferred to a nitrocellulose membrane for immunoblotting. For neutrophils, their intrinsic protease activity prevents quantification of protein content, as samples are significantly degraded in that time. Therefore, neutrophils were counted to obtain equal neutrophil, were flash frozen in liquid nitrogen and lysed directly in 1X Laemmli buffer for protein separation by SDS-PAGE. Antibodies used were anti-MCL1 (Cell Signaling), anti-BCL2 (Cell signaling), anti-eIF4E (BD Biosciences), anti-phospho-eIF4E clone EP2151Y (Abcam), anti-β-actin clone AC15 (Sigma), anti-ERK (Santa Cruz), anti-phospho-ERK (Cell Signaling), anti-4E-BP1 (Cell Signaling).

Quantification and statistical analysis All statistical analysis was performed using GraphPad Prism software. All graphs, except those presenting fractions of entire populations such as Kaplan-Meier curves, are shown as means +/- SEM. Significance was defined using the standard p=0.05 cut-off. P-values, and the values and meanings of n can be found in the figures and legends. Statistical tests used are outlined below. Unpaired t-test. For comparisons of only two groups without paired samples, the unpaired t-test was used, as was the case for Figure 3.6C-E, where there was no indication that populations deviated significantly from a normal distribution or had widely different variations.

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2-way ANOVA. Several experiments were performed where a variety of measurements were taken in two groups, including figures 3.3B, 4ABD, 5ABD and 6F; these were analyzed by two-way ANOVA. Some p-values are indicated for pair-wise comparisons of interest and were computed using Sidak’s multiple comparison test. Repeated measures ANOVA. For experiments monitoring tumor growth and neutrophil accumulation in circulation, several measurements at different time points were performed on the same animals; thus, repeated measures ANOVA was used in figures 3.2B, 3D, 4C and 6B. Fisher’s exact test. For experiments in which data could be categorized as a 2X2 contingency table, Fisher’s exact test was used. This is the case in the following figures: Figure 3.1B, where all samples contained either normal epithelial cells or neoplastic epithelial cells that expressed either high or low levels of eIF4E phosphorylation; Figure 3.1C, where all samples contained either normal epithelial cells or neoplastic epithelial cells, and were either positive or negative for eIF4E phosphorylation in cells of the surrounding stroma; Figure 3.2E, where metastases were either intra-vascular or extra-vascular and present in either WT or eFI4ES209A mice; Figure 3.5C, where neutrophils where either segmented or apoptotic and present in either WT or eFI4ES209A mice.

3.5 ACKNOWLEDGEMENTS This work was by supported by The Susan G. Komen Breast Cancer Foundation (IIR12224057), CIHR (MOP-7214) and the Canadian Cancer Society (2010-700377) to N.S. W.H.M. (2012-17280) and P.M.S. (2015-20042) were supported by grants from the Cancer Research Society. M.P. holds funds from the FRQS (FRQ-34787) and Quebec Breast Cancer Foundation. We thank the Histology Facility at the Goodman Cancer Research Centre for tissue processing, the Cell Vision Facility for flow cytometry and the Comparative Medicine and Animal Resources Centre for complete blood counts and differentials. The authors have no conflicts of interest to declare.

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CHAPTER 4

GENERAL DISCUSSION

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4.1 How does eIF4E phosphorylation affect translation?

In the current thesis, the importance of eIF4E phosphorylation in cancer cells and neutrophils is established. However, it remains unclear how eIF4E phosphorylation affects the translation of specific mRNAs at the molecular level. Many models have been proposed, one of which has been introduced briefly in Chapter 1; these are discussed in greater detail here, in light of the data presented in Chapters 2 and 3.

4.1.1 Switch to alternate initiation mechanisms

The phosphorylation of eIF4E has been proposed to inhibit cap-dependent translation while promoting cap-independent translation via IRESs (465). This hypothesis is supported by the finding that eIF4E phosphorylation reduces its affinity for the cap, thus purportedly favoring cap-independent translation (59-61). Such a mechanism would be particularly useful in stress conditions that inhibit mTOR but activate p38 MAPK signaling, such as hypoxia (466). In line with this, eIF4G upregulation has been shown to promote IRES-mediated translation of Vegfa when 4E-BPs are hypophosphorylated in response to hypoxia (191). Furthermore, MNK activity promotes cap-independent translation by the Myc IRES in multiple myeloma cells treated with rapamycin (465). Similarly, MNKs promote the translation of oncolytic poliovirus mRNAs containing IRES sequences (467), although similar effects have also been attributed by the same group to other targets of the MNKs (468).

Unfortunately, this is a recurring theme in studies on the effects of eIF4E phosphorylation: as its modulation is most easily achieved by MNK inhibition or knockdown, most studies rely exclusively on these methods, and attribute the reported effects to the best characterized substrate, eIF4E. However, the design of these studies cannot distinguish between MNK targets including eIF4E (469), Sprouty2 (393), cPLA2 (394) and hnRNPA1 (395, 396). As a result, several effects ascribed to eIF4E phosphorylation have subsequently been attributed to other effectors, including translational regulation of TNFα and the afore-mentioned regulation of cap-independent translation in virus-infected cells (396, 468).

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In contrast, the work presented here unambiguously attributes pro-metastatic functions to translational control via eIF4E phosphorylation. While we did not specifically investigate IRES-mediated translation, our results fail to uncover any evidence of increased cap- independent translation. Thus, polysome profiling of S209A MEFs did not detect any change in the translation of the Myc mRNA, or other commonly reported IRES-bearing mRNAs such as Vegfa or Bcl2 (55). Of note, these experiments were performed in optimal growth conditions where cap-dependent translation is predominant, whereas previous reports were performed in the context of mTOR inhibition, perhaps explaining the discrepancy. While decreased expression of BCL2 in S209A neutrophils was detected in Chapter 3, we cannot attribute this effect to impaired IRES-mediated translation, as it could result from the blockade of either cap-dependent or -independent translation. Thus, while a phospho-eIF4E dependent switch to IRES-mediated translation is not required to explain our results, it cannot be wholly excluded.

Phosphorylation of eIF4E has also been reported to control the identity of the major cap- binding protein by promoting the translation of the Eif4e mRNA, with MNK inhibition resulting in the translation of the alternative cap-binding protein eIF4E3 (470). Thus, phospho-eIF4E was proposed to promote oncogenic cap-dependent translation, in opposition to the supposed tumor-suppressive program of eIF4E3-mediated cap-dependent translation (471). While eIF4E3 levels were not assessed in any of our experiments, eIF4E levels were routinely monitored in cells lacking MNKs, in cells bearing the S209A mutation, and in response to MNK inhibition; as a result, we are confident that eIF4E levels are not affected by its phosphorylation in any cells tested, including neutrophils, breast and prostate cancer cell lines, B16 melanoma cells, primary MEFs, transformed MEFs, and mouse mammary tumors. We cannot, however, exclude that such an effect exists specifically in DLBCL.

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4.1.2 Modulating protein-protein interactions One possible mechanism by which phosphorylation of eIF4E may affect translation is by altering its affinity for certain interacting proteins. This has been reported as the mechanism underlying increased migration and invasion downstream of eIF4E phosphorylation. Thus, eIF4E phosphorylation reduces its affinity for the repressor of translation cytoplasmic fragile- X protein-interacting protein 1 (CYFIP1), resulting in increased availability for cap-dependent translation (422). In contrast, phospho-eIF4E has been proposed to increase its affinity for the eIF4E transporter 4E-T, leading to its recruitment to mRNP granules, in response to a variety of stressors (233). As both of these reports were published subsequent to our own work presented in Chapter 2, neither mechanism was investigated in the experiments presented therein. Nonetheless, it is unclear how phosphorylation of eIF4E would affect its affinity for its known interacting proteins, as S209 is present on a flexible loop near the cap- binding channel, opposite the canonical binding site for eIF4G, the 4E-BPs, CYFIP1 and 4E-T. While secondary binding sites nearer S209 have been reported for eIF4G and the 4E-BPs (50, 51), the effect of phosphorylation on this region remains unclear. Indeed, there is as of yet no structural information on the phosphorylated form of eIF4E.

In contrast, eIF4E-binding partners may influence eIF4E phosphorylation by the MNKs. Indeed, MNK1 must be recruited by eIF4G to phosphorylate eIF4E (54). Thus, binding of any other interactor would prevent MNK1 recruitment and eIF4E phosphorylation, which has been demonstrated in the case of 4E-BPs binding to eIF4E as a result of mTOR inhibition (472). However, it remains to be established whether other eIF4E interacting proteins can recruit MNKs in certain conditions. It is also unclear whether any intermediates are required for phosphorylation of eIF4E by MNK2; while MNK2 can bind eIF4G, studies establishing the requirement for eIF4G have focused on MNK1 (54, 410, 473). MNK2 also displays higher basal activity and is relatively independent of activation from upstream signaling (410, 474). There is as-of-yet unpublished data supporting the hypothesis of eIF4G-independent phosphorylation of eIF4E by MNK2 (Graff JR, personal communication). As a result, there may be unsuspected mechanisms of eIF4E phosphorylation that are independent of eIF4G

111 and its proposed role in promoting initiation factor recycling after 43S PIC recruitment. If correct, this may partially explain why some phenotypes associated with eIF4E phosphorylation appear to be MNK1 dependent while others appear to be MNK1 dependent, as observed in studies distinguishing between individual eIF4E kinases (for example: (97, 298, 392, 474)).

One interesting possibility that has yet to be investigated is that eIF4E phosphorylation may play a role in substituting cap-binding proteins in mRNP granules. Indeed, phospho-eIF4E has been reported to play a role in the stress response and to be elevated in P-bodies and/or stress granules (233, 475). In parallel, it has been suggested that eIF4E may be displaced from the cap in conditions of miRNA-mediated silencing in favor of the alternative cap- binding protein 4EHP (476). While there is currently no data to support this idea, it may be worth speculating that phosphorylation of eIF4E could reduce its affinity for the cap, thus promoting its replacement by 4EHP. Of course, this depends on the possibility that eIF4E can be phosphorylated independently of the eIF4F complex for processes other than translation initiation. Testing this hypothesis should be straightforward, for example by repeating the experiments contained in reference (476), but in cells and lysates lacking the MNKs or with recombinant eIF4E bearing the S209A mutation.

4.1.3 Promoting initiation factor recycling after 43S PIC recruitment As described in section 1.4.3, one model proposes that eIF4E phosphorylation decreases its affinity for the cap and occurs after scanning has begun to promote initiation factor recycling (63). Thus, phosphorylation of eIF4E is hypothesized to have similar effects to increased eIF4E availability or expression, an idea supported by the fact that all three of these contexts increase the translation of mRNAs containing stable hairpins in their 5’UTRs (13, 64). While not explicitly tested, several reports support this model. When ribosomes and their interacting proteins are separated from other cytoplasmic proteins in rabbit reticulocyte lysates by centrifugation or chromatography, the fraction of eIF4E bound to ribosomes is phosphorylated at higher proportion (~60%) than “free” eIF4E (~30%) (477). Furthermore,

112 the majority of eIF4E is unbound. These findings support the idea that phosphorylation occurs following ribosome recruitment, and results in eIF4E release, consistent with early reports that eIF4E binds to 48S initiation complexes but not 80S ribosomes (478). Conceptually, this would allow for eIF4E to participate in the formation of new 48S complexes during AUG recognition and 60S subunit joining, a particularly slow process, as shown by the accumulation of 40S and 80S ribosomes at start codons (479).

In addition, dephosphorylation of eIF4E must be relatively rapid to prevent accumulation of phosphorylated eIF4E in the cytoplasm. While phosphatase activity is generally assumed to be faster than that of kinases, the identity of the eIF4E phosphatase remains uncertain; it thus remains to be demonstrated whether the kinetics of its dephosphorylation are sufficient to support the proposed model of increased initiation factor recycling. Encouragingly, several phosphatases in the MAPK pathway display high catalytic efficiencies, in the range of 106 M-1s-1, 1000-fold greater than that of their respective kinases (480). While eIF4E dephosphorylation has not been thoroughly investigated, protein phosphatase 2A (PP2A) has been reported to be responsible for dephosphorylation of both MNK1 and eIF4E (481). However, it has also been reported to dephosphorylate 4E-BP1, thus, eIF4E may be a direct PP2A target or be indirectly regulated by controlling eIF4F complex formation and MNK activity (482). Interestingly, the B56 regulatory subunit of PP2A has been identified as a possible interactor with eIF4G (483). If correct, this finding would implicate the presence of an eIF4E phosphatase in close proximity with its substrate, allowing for rapid and dynamic phosphorylation and dephosphorylation, such as would be required for the initiation factor recycling model.

Although our results cannot answer this issue, they highlight important differences in the kinetics of phosphorylation and dephosphorylation of signaling intermediates upstream of eIF4E. Thus, MAPK signaling increases by 3 hours of TGFβ stimulation and returns to baseline levels by 14 hours; in contrast MNK1 and eIF4E phosphorylation remain elevated for at least 24 hours. This suggests that the phosphatases responsible for the dephosphorylation of

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MNKs are distinct from those responsible for upstream kinases. For the MNKs, the phosphatase likely displays only a low level of expression and/or activity, as its phosphorylation remains high despite reduced activity of its upstream kinase. The activity of the eIF4E phosphatase likely remains constant, as eIF4E phosphorylation remains at a constant, increased level, mirroring that of its activated kinase. We believe that the phosphorylation of eIF4E is unlikely to simply achieve saturation caused by hyperactive MNKs, as even their most potent activators result in only about 75% of eIF4E present in the phosphorylated form (484).

Of note, the finding that TGFβ stimulation results in the translational activation of mRNAs transcriptionally induced by TGFβ highlights another potential role of eIF4E phosphorylation: recruitment to new mRNAs. Indeed, it is possible that, in unstimulated cells, eIF4E is mostly bound to mRNAs required to maintain their homeostasis. Upon stimulation, eIF4E must be redirected to new mRNAs needing to be translated to respond to the stimulus. Phosphorylation may be a means to release eIF4E from mRNAs previously undergoing translation to promote the translation of newer ones. The basis for this idea was proposed as part of the initiation recycling hypothesis, and has yet to be thoroughly tested (63).

In fact, all aspects of how eIF4E phosphorylation affects translation may need to be revisited. While there have been many studies on this topic, the design and conclusions of the experiments may have been biased by unrealistic expectations skewed by an unfortunate historical error. Indeed, the phosphorylation site of eIF4E was initially misidentified as serine 53 (485). Eventually, the correct site was identified by the same group and independently confirmed (486, 487), but in the meantime, various studies using an S53A mutant had concluded that eIF4E phosphorylation was an absolute requirement for translation initiation (98, 488-491). As a result, subsequent studies on S209 were characterized by statements that its phosphorylation was “not required” for a variety of processes, including functional replacement of yeast eIF4E, the reconstitution of eIF4E-depleted rabbit reticulocyte lysates, cell growth, organismal development, or de novo protein synthesis following hypertonic

114 stress (474, 492, 493). However, revisiting the data presented in these reports tells a different story. First, it is not surprising that phosphorylated S209 plays no role in Saccharomyces cerevisiae, as yeast do not possess an ortholog of MNK1 or residues equivalent to S209 in the eIF4E ortholog. In contrast, flies, which can phosphorylate deIF4E via their MNK1 ortholog, display growth defects in response to amino acid deficiency (494). Second, while the data presented in (493) shows that reconstitution of eIF4E-depleted rabbit reticulocyte lysate with high levels of eIF4E S209A is equivalent to reconstitution with WT eIF4E, it is clearly less effective at intermediate levels. Similar observations can be made regarding de novo protein synthesis, as small molecule MNK inhibition does not entirely prevent polysome formation following hypertonic stress, but clearly reduces its efficiency (492). Thus, it may be useful to further investigate the requirement of eIF4E phosphorylation for the initiation of translation by performing reconstitution experiments with increasing amounts of eIF4E mutants, or by testing de novo protein synthesis following various stresses in cells lacking MNKs or expressing eIF4E S209A. A role for eIF4E phosphorylation in directing initiation factors to newly synthesized mRNAs could also be tested by controlling the sequential addition of in vitro synthesized reporter mRNAs in rabbit reticulocyte lysates reconstituted with eIF4E mutants.

Clearly, more work is needed to dissect the molecular effects of eIF4E phosphorylation, its impact on translation, and potentially other cellular processes. Curiously, many of the competing hypotheses described above may contain some truth, as they are not mutually exclusive. Changes in affinity for the cap and binding partners may occur simultaneously, as can changes in recycling and subcellular localization. There may even be different contexts during which eIF4E phosphorylation may be dynamic and promote recycling, or continuously elevated and reduce cap binding and cap-dependent translation.

4.2 Revisiting the pro-metastatic effect of eIF4E phosphorylation in cancer cells The experiments presented herein demonstrate that translational control plays an important role in the metastatic process, both in cancer cells and in the cells of the TME. Specifically,

115 we demonstrate that eIF4E phosphorylation promotes the translation of mRNAs in different cell types that promotes their pro-metastatic functions. In neutrophils, this favors their survival in response to G-CSF by promoting the translation of mRNAs encoding the anti- apoptotic factors BCL2 and MCL1. In cancer cells, invasion is favored by promoting the translation of mRNAs encoding SNAIL and MMP3, two factors that promote EMT. Furthermore, we validate some of these findings in vivo, by demonstrating that neutrophil survival in response to eIF4E phosphorylation leads to aberrant accumulation and favors lung colonization by cancer cells in an orthotopic model. We also used a genetic model that confirms that tumors expressing phospho-eIF4E are more metastatic and express more SNAIL and MMP3. However, the conclusions drawn from the genetic experiments in Chapter 2 should be tempered by our findings in Chapter 3. Indeed, in the MMTV-PyMT model, the S209A mutation is present in all cell types of the eIF4ES209A mice, including neutrophils. Thus, we cannot exclude that the observed difference in metastatic potential between WT and S209A mice is due to differential phospho-eIF4E-dependent neutrophil survival, rather than phospho-eIF4E-dependent cancer cell invasion. Considering that neutrophils have been described to play an important role in the metastatic dissemination of MMTV-PyMT tumors (495), our inability to separate the contributions of the cancer cells from those of the neutrophils is an important caveat.

In an attempt to address this issue, we have devised several plans to use orthotopic models in which we can individually control eIF4E phosphorylation in the tumor and the host. Unfortunately, these have so far been hampered by significant technical limitations. In one case, WT and eIF4ES209A tumors were isolated, dissociated and reinjected into naïve recipient mice of both genetic backgrounds. We tested the four possible combinations of phospho- eIF4E in the cancer cells only, the TME only, both compartments or neither, in an effort to dissect the relative contribution of each. While some of our other findings were validated, notably that eIF4E phosphorylation in cancer cells, but not the TME, promotes the onset of mammary tumors, none of the mice developed metastatic lung colonies, preventing any further analysis. This was surprising, considering the high metastatic burden observed in

116 almost all mice in the genetic MMTV-PyMT model. The reduced lung colonization is likely due to a reduced tumor load in the orthotopic model, where mice bear a single primary tumor, as opposed to 10 or more in the genetic model. Alternatively, we endeavored to abrogate eIF4E phosphorylation in 66cl4 cells to compare their metastatic potential with the parental line. First, we used shRNAs targeting MNK1/2, with the objective of isolating clones with stable knockdown of these kinases. However, MNK expression invariably was recovered within one week, preventing their use in vivo, where tumors form over 4-5 weeks. We are currently circumventing this problem by using CRISPR-CAS9 gene editing to knock out the MNKs, and are awaiting the identification of successfully mutated clones. While experiments using these cells will be crucial in confirming that eIF4E phosphorylation in cancer cells plays a role in metastasis in vivo, our previously obtained in vitro data indicating that phospho- eIF4E promotes EMT and invasion remains valid.

4.3 Other inhibitors of translation in immune space In this thesis, emphasis was entirely placed on the phosphorylation of eIF4E as a model for translational control. However, many other regulatory events in protein synthesis may be important in the metastatic process. In fact, considering the relative innocuousness of the eIF4ES209A mutation, other targets may provide even greater benefits. Thus, the Pelletier lab has demonstrated that eIF4F complex formation is critical in cancer cell dissemination, and that its pharmacological targeting using inhibitors of mTOR or eIF4A efficiently prevents metastasis (237). However, nothing is known regarding the effect of eIF4A inhibition in neutrophils or any other immune cell population, although it is potently cytotoxic in Jurkat cells and various lymphoma and leukemia models (496, 497).

More is known regarding how mTOR inhibitors affect the tumor microenvironment, as they are well documented to be immunosuppressive. Thus, mTOR inhibitors prevent T cell growth and proliferation, while promoting Treg development, resulting in impaired adaptive immunity (373, 498). Rapamycin, in particular, has been used as a clinical immunosuppressive for decades. Interestingly, one model demonstrates the importance of

117 mTOR in both cancer cells and TME: renal transplant recipients that develop Kaposi’s sarcoma. Indeed, these tumors can appear in patients receiving cyclosporin to prevent graft rejection. Interestingly, substituting rapamycin for cyclosporin maintains immunosuppression by inhibiting mTOR in T cells, but also leads to tumor regression by inhibiting mTOR in cancer cells (499, 500). Other reports indicate that mTOR regulates the accumulation of myeloid-derived suppressor cells (MDSC) in transplant and allograft recipients, although it is unclear whether this is due to effects intrinsic to MDSCs, or whether this is secondary to effects in other cell types (377-379).

Another targetable branch of translation is the eIF2α pathway, with small molecules being developed that can increase or reduce eIF2α phosphorylation, or inhibit TC recruitment to the PIC. These compounds show promise in treating leukemias and other cancer types by inhibiting translation initiation and causing apoptosis (102, 138-140, 314, 338), but their effects on non-transformed immune cell types remains to be determined. One study exceptionally investigated the effect of PERK inhibition on immune cells, in particular in the brain microenvironment, and found that normal immune function remained intact, while ER- stress induced inflammation was reduced (501). Another study has reported that guanabenz- induced increases in eIF2α phosphorylation inhibits the expression of GM-CSF, IL6 and COX2, which may be beneficial (502). The effect of such therapeutic strategies on both the tumor and its microenvironment remains to be investigated. Of note, eIF2α phosphorylation was shown to play an important role in separating T helper cell priming from activity by preventing the translation of mRNAs encoding inflammatory cytokines at the priming site and in circulation; their translation at the target site occurred following a second T cell activation event, resulting in eIF2α dephosphorylation (503). It is thus possible that targeting the eIF2 pathway in patients would affect T cell mobilization in the cancer setting, but how and to what extent remains to be seen.

The importance of translation in T cells is especially relevant to the cancer setting, as these are considered the major cell type in controlling tumor progression, and are the target of

118 immunotherapeutic drugs currently providing significant benefits to cancer patients that had previously lacked useful therapeutic options. Interestingly, the expression of initiation factors increases in activated T cells over that of quiescent T cells to support the increased protein synthesis associated with their proliferation (504, 505). In contrast, eIF4E levels and mTOR activity decrease in T regulatory cells (Tregs), and the resulting reduction in translation is required for their differentiation (380, 498). Considering these findings, great care must be taken when utilizing inhibitors of translation to determine their effects on the immune system. For example, if an inhibitor of mTOR causes apoptosis in cancer cells and prevents the accumulation of MDSCs, but simultaneously reduces CD8+ T cell proliferation and activity while increasing Treg differentiation, the net outcome is difficult to predict. It may, in fact, be undesirable if the balance favors immunosuppression over cancer cell killing. Biomarkers should be identified that can predict which effect will be dominant to determine which patients would benefit most from mTOR inhibitors, for example, levels of circulating MDSCs, presence and activity of infiltrating T cells, etc. Alternatively, targets with beneficial effects in both cancer cells and the immune system could be prioritized. Such is the case for the phosphorylation of eIF4E, as presented here, but other, yet-to-be-identified targets likely exist.

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Conclusion and perspectives The work presented in this thesis highlights the importance of translation in cancer. First, it identifies the phosphorylation of eIF4E as a key event promoting the translation of pro- metastatic mRNAs in cancer cells. Second, it expands the importance of eIF4E phosphorylation to not only cancer cells, but also non-transformed cells of the tumor microenvironment, by demonstrating its role in promoting the survival of neutrophils in response to G-CSF. Overall, the findings described herein underscore a need to study the effects of inhibitors of translation currently being developed on both cancer cells and the TME.

As we ponder the sometimes overwhelming complexity of translational deregulation in cancer, we are left wondering which aspects will prove most vulnerable and amenable to therapeutic intervention. eIF2A certainly appears to be a promising target, though the knowledge of its potential is too recent for drug discovery to be in anything but its infancy. Certainly, there is much interest in the development of eIF4A inhibitors, as well as several other translation initiation factors. Beyond the potency of such drugs on cancer cells, several questions will need to be addressed. What will their effects be on other cell types of the TME? What are the possibilities for cancer cells to develop resistance to such inhibitors? While downstream effectors of oncogenic kinases can easily be identified, overexpressed, constitutively activated or phosphorylated by alternative pathways, it is unclear what equivalent process could circumvent the inhibition of translation. Can feedback inhibition limit the efficacy of targeting translation, as seen with kinase inhibitors? While several positive feedback loops have been described, most notably the interplay between MYC and the translational machinery, examples of negative feedback, while existent, have been largely ignored by the field. Certainly, it is an exciting time to study the translational control of cancer.

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APPENDIX A SUPPLEMENTAL TABLES AND FIGURES

Supplemental Table 1.1: Abbreviations

Abbreviation Explanation eIF… Eukaryotic translation initiation factor… followed by numbers and letters for individual identification (as in eIF3, eIF4E, etc.) PIC Pre-initiation complex containing 40S ribosome and eIFs 4E-BP eIF4E-binding protein, followed by a number to distinguish homologs or by “s” to refer to several (e.g. 4E-BP1, 4E-BPs) PDCD4 Programmed cell death 4 MNK MAPK integrating kinase MAPK Mitogen-activated protein kinase RNA Ribonucleic acid mRNA Messenger RNA rRNA Ribosomal RNA tRNA Transfer RNA Met-tRNAi Methionine initiator tRNA TC Ternary complex composed of eIF2, GTP, Met-tRNAi miRNA Micro RNA UTR Untranslated region preceding (5’) or following (3’) the ORF ORF Open reading frame, the section of mRNA encoding a protein uORF Upstream ORF, an ORF situated 5’ of the main ORF mTOR Mammalian target of rapamycin mTORC1 mTOR complex 1, sensitive to rapamycin asTORi Active-site mTOR inhibitor S6K Ribosomal protein S6 kinase Phospho- The phosphorylated form of a protein PABP Poly-A binding protein IRES Internal ribosome entry site VEGFA Vascular endothelial growth factor A BCL2 B-cell lymphoma 2, an anti-apoptotic protein MCL1 Myeloid cell leukemia 1, an anti-apoptotic protein ATF4 Activating transcription factor 4, a stress-response factor MYC V-Myc Avian Myelocytomatosis Viral Oncogene Homolog HIF1α Hypoxia-inducible factor 1-alpha IFNγ Interferon gamma, a cytokine TNFα Tumor necrosis factor alpha, a cytokine TGFβ Transforming growth factor beta, a cytokine MDSC Myeloid derived suppressor cell TME Tumor microenvironment

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Supplemental Table 1.2: Dysregulation of translation initiation factors in human cancers

Factor Dysregulation Clinical correlates in cancers eIF4E Overexpression Decreased survival in breast (506), head and neck (507), liver (508), prostate (116), bladder (509), stomach (510). Correlates with disease progression and aggressive subtypes in many cancers (133, 134, 511-514) and resistance to chemotherapy (511) eIF4E Phosphorylation Elevated in early stages of development of breast, colon, gastric, and lung cancers (115). Increased in prostate cancer and correlates with androgen independence (55, 116). Poor prognosis marker in non-small cell lung cancer (386) 4E-BP1 Overexpression Inversely correlates with tumor grade (282, 513) Correlates with better survival in lung and prostate cancers (116, 515) Correlates with absence of lymph node and distant metastases in gastric cancer (516) 4E-BP1 Loss Possibly responsible for loss of translational control in 50% of pancreatic tumors (280) 4E-BP1 Phosphorylation Correlates with tumor grade and poor prognosis in breast, lung, ovarian and prostate cancers (116, 282, 517-519) eIF4G Increased Amplification correlates with aggressive stages in lung expression cancer (188). Overexpressed in inflammatory breast cancer (108) and cervical cancer (520). Correlates with poor prognosis in nasopharyngeal carcinoma (521). eIF4A Increased Overexpressed in lung (188) and cervical cancer (520). expression Lowered expression after radiation predicts better survival in cervical cancer (520). PDCD4 Decreased Associated with poor prognosis in breast (121), lung (522), expression colon (523) and ovarian cancers (123) and gliomas (524). Inversely correlated with advanced tumor stage in RCC (525) eIF2α Increased Correlates with aggressive lymphoma subtypes (133). expression eIF5A Increased Correlates with poor prognosis in early-onset colorectal expression and cancer (526). Overexpression of eIF5A2 correlates with local hypusination invasion in NSCLC (527) and HCC (528).

158 eIF6 Altered Elevated in colorectal cancer (168), head and neck carcinomas expression and (169) and ovarian serous carcinoma (529); low expression function correlates with reduced disease-free survival in ovarian serous carcinoma (529); mediates lymphomagenesis in Shwachman-Diamond syndrome (171, 530, 531) eIF3a Increased breast (532), cervix (533), esophagus (534), lung (535), expression stomach (536) eIF3b Increased bladder (537), breast (538), prostate (537) expression eIF3c Increased meningioma (539), testicular seminoma (540) expression eIF3h Increased breast (541), colon (542), liver (543), prostate (541, 544) expression eIF3i Increased breast (545), head and neck (546), liver (547), melanoma expression (548), neuroblastoma (548) eIF3m Increased colon (549) expression eIF3e Decreased breast (550), lung (551), prostate (550) expression eIF3f Decreased breast (552), colon (552), small intestine (552), melanoma expression (553), ovary (552), pancreas (554), vulva (552)

Figure S2.1. YB1 cannot explain invasion differences. Western blot analysis of indicated proteins in WT and Myc/Ras-transformed WT and eIF4ES209A MEFs.

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Figure S2.2. Clustered pathways of eIF4E phosphorylation regulated mRNAs. Screenshots of the complete output of clustering from the DAVID software of significantly enriched pathways from a previously generated list of phospho-eIF4E translationally-regulated mRNAs.

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Figure S2.3. Complete list of mRNAs clustered by the DAVID software.

Figure S2.4. Non phospho-eIF4E regulated mRNAs involved in EMT/invasion and metastasis. qRT-PCR analysis of polysomal fractions of (A) Twist (B) MPP14, (C) vimentin, in Myc/Ras- transformed WT and eIF4ES209A MEFs. Concentrations of mRNA in each fraction are normalized to GAPDH. Error bars represent standard deviations of each fraction. Results are representative of at least 2 experiments.

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Figure S2.5. eIF4E phosphorylation does not affect Snail mRNA levels. qRT-PCR analysis of Snail mRNA in NMuMG cells treated with 5ng/mL TGFβ, (A) with or without 20μM CGP57380 or (B) in the presence of scramble siRNA or siMNK1.

Figure S2.6. Inhibition of eIF4E phosphorylation maintains tight junctions during TGFβ induced EMT. Quantification of ZO1 immunofluorescence in NMuMG cells treated with 5ng/mL TGFβ, (A) with or without 20μM CGP57380 or (B) in the presence of scramble siRNA or siMNK1.

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Figure S2.7. eIF4E phosphorylation correlates with EMT inducers and metastatic potential in vivo. Western blot analysis of eIF4E phosphorylation and transcription factors that induce EMT in tumor lysates formed by indicated isogenic cell lines.

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Figure S3.1. Scoring and histopathology of breast cancer samples. (A) Examples of phospho- eIF4E scoring in the epithelial cells and TME. Phospho-eIF4E expression in cancer cells was categorized as “low” if undetectable or if expression was low and sparse; it was categorized as “high” if expression was uniformly detectable or if expression was sparse but very high. Phospho- eIF4E expression in the TME was categorized as “positive” if at least 5 cells outside the tumor bed expressed phospho-eIF4E, otherwise it was categorized as “negative”. Shown are examples of a tumor where expression in cancer cells was low and negative in the TME (top left), low in the cancer cells and positive in the TME (top right), high in the cancer cells but negative in the TME (bottom left), high in the cancer cells and positive in the TME (bottom right). (B-F) Clinical correlates of samples stratified by phosphorylation of eIF4E in the TME; no statistical analysis is provided as the sample numbers do not provide the power to perform the multiple tests required. Show are tumor size (B), patient age at biopsy (C), hormone receptor status (D), tumor grade (E) and lymph node status (F). Not all information was available for all samples. For hormone receptor status, some samples are counted more than once if positive for multiple receptors. N/D not determined

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Figure S3.2. Similarities between primary tumors and healthy immune systems of WT and eIF4ES209A mice. (A) Quantification of CD31, ki67 and cleaved caspase 3 in tumors from WT and eIF4ES209A mice from immunohistochemical staining as in Figure 3.1C. (B) Tumor growth and lung metastatic burden of C57/BL6 mice of the indicated genotype bearing tumors derived from EO771 cells. (C-G) Spleens of WT and eIF4ES209A mice were dissociated and by flow cytometry using the indicated markers. (B) Quantification of monocytes and neutrophils based on Ly6C and Ly6G expression. (C) Quantification of T cells and B cells based on CD3 and CD19 expression. (D) Quantification of T cell subtypes based on CD4 and CD8 expression. (E-F) T cells isolated from WT and eIF4ES209A spleens were incubated with anti-CD3 to stimulate cytokine production, followed by flow cytometric analysis of TNFα (E) and IFNγ (F).

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Figure S3.3. T cells and NK cells are unlikely candidates to mediate the effects of eIF4E phosphorylation on the TME. (A) Primary tumors were dissociated and single cell suspensions were analyzed by flow cytometry using markers for cytotoxic T cells and regulatory T cells, based + + on the expression of CD3, CD4, CD8 and FOXP3. The ratio of CD8 T cells to FOXP3 Tregs is presented in (A). (B-C) 66cl4 cells were injected into the fat pad of WT BALB/c mice and BALB/SCID mice. No differences were observed in tumor growth (B) or metastasis to the lung (C). (D) NK cell-mediated protection from lung colonization was assayed in WT and eIF4ES209A mice: NK cells were depleted by intraperitoneal injections of anti-ASIALO-GM1 2 days prior to tail vein injections of 66cl4 cells, on the day of injection and every other day for the following 12 days. The relative increase in lung colonization from control-treated to NK cell-depleted mice was compared between WT and eIF4ES209A mice (D). (E-F) Neutrophil depletion with Ly6G antibodies had no effect on levels of circulating monocytes (E) and lymphocytes (F), as determined in complete blood counts using an automated analyzer.

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Figure S3.4. Neutrophil function and differentiation. Polarization of high density neutrophils (HDN) to pro-metastatic low density neutrophils (LDN) by the serum of tumor-bearing mice was assessed (A). Briefly, HDNs and LDNs from WT and eIF4ES209A mice were isolated by density gradient centrifugation, stained with CD11b and Ly6G and sorted using a BD Aria Fusion flow cytometer. HDNs were stained with CFSE and incubated for 4 hours in whole blood from WT tumor-bearing mice. The resulting cell population was separated again by density gradient centrifugation and the proportion of CFSE positive cells in the HDN and LDN populations was assessed by flow cytometry. In addition, FACS sorted HDNs and LDNs, as in (A), from n=4 mice, were incubated in the presence of CFSE-stained WT and eIF4ES209A T cells, and T cell proliferation was induced by incubation with recombinant CD3/CD28 for 3 days. T cell proliferation was then quantified by flow cytometric analysis of CFSE content: the proportion of T cells in the fastest dividing population (P7) is quantified in (B). Phosphorylation of eIF4E in both neutrophils and T cells had no significant effect of T cell proliferation. (C) Gelatin zymogram depicting MMP9 expression in HDNs and LDNs from WT and eIF4ES209A mice. (D) Lin-/Sca+/cKit+ cells were isolated from the bone marrow of naïve mice and differentiated in vitro in the presence of IL3, SCF and G-CSF; neutrophil differentiation and viability was monitored by flow cytometric analysis of Ly6G/Ly6C and 7-AAD (E-F).

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